Method for transposase mediated spatial tagging and analyzing genomic dna in a biological sample

ABSTRACT

The present disclosure relates to materials and methods for spatially analyzing nucleic acids fragmented with a transposase enzyme, optionally complexed to an antibody-binding moiety (e.g., an antibody-binding protein) bound to an antibody for at least one chromatin protein, in a biological sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation to U.S. patent application Ser. No.18/074,081, filed on Dec. 2, 2022, which claims priority to U.S.Provisional Patent Application No. 63/285,677, filed Dec. 3, 2021. Theentire content of the foregoing application is incorporated herein byreference.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submittedelectronically as an XML file named 47706-0322002_SL_ST26.xml. The XMLfile, created on May 4, 2023, is 3,772 bytes in size. The material inthe XML file is hereby incorporated by reference in its entirety.

BACKGROUND

Cells within a tissue have differences in cell morphology and/orfunction due to varied analyte levels (e.g., gene and/or proteinexpression) within the different cells. The specific position of a cellwithin a tissue (e.g., the cell's position relative to neighboring cellsor the cell's position relative to the tissue microenvironment) canaffect, e.g., the cell's morphology, differentiation, fate, viability,proliferation, behavior, signaling, and cross-talk with other cells inthe tissue.

Spatial heterogeneity has been previously studied using techniques thattypically provide data for a handful of analytes in the context ofintact tissue or a portion of a tissue (e.g., tissue section), orprovide significant analyte data from individual, single cells, butfails to provide information regarding the position of the single cellsfrom the originating biological sample (e.g., tissue).

Chromatin structure can be different between cells in a biologicalsample or between biological samples from the same tissue. Assayingdifferences in accessible chromatin can be indicative oftranscriptionally active sequences, e.g., genes, in a particular cell.Exemplary methods for assaying differences in accessible chromatininclude, but are not limited to, nucleosome occupancy and methylomesequencing (NOMe-seq), assay for transposase-accessible chromatin usingsequencing (ATAC-seq), DNase I hypersensitive sites sequencing(DNAse-seq), Hi-C sequencing, RNA-seq, bisulfate sequencing (BS-seq),DNA modification-dependent restriction endonuclease AbaSI coupled withsequencing (Aba-seq), chemical-labeling-enabled C-to-T conversionsequencing (CLEVER-seq), chromatin immunoprecipitation with massivelyparallel sequencing (ChIP-seq), cleavage under targets and release usingnuclease (CUT & Run), cleavage under targets and tagmentation (CUT &Tag), scCUT & Tag, high-spatial-resolution chromatin modification stateprofiling by sequencing (hsrChST-seq), and single cell sequencingvarieties thereof. Further understanding the transcriptionally activeregions within chromatin will enable identification of which genescontribute to a cell's function and/or phenotype.

SUMMARY

The present disclosure generally describes methods for spatiallyanalyzing genomic DNA present in a biological sample.

Methods have been developed to study epigenomes, e.g., CUT & Run or CUT& Tag sequencing. These assays help identify regulators (e.g., cisregulators and/or trans regulators) that contribute to dynamic cellularphenotypes. While CUT & Run and CUT & Tag sequencing have been valuablein defining epigenetic variability within a cell population,conventional applications of these methods are limited in their abilityto spatially resolve the two- and three-dimensional structures andassociated genes that promote cellular variation. Although spatialmethods are also known, additional and/or alternative methods are stillneeded. In particular, methods that can simultaneously assess epigenomesand gene expression in a tissue sample would be useful.

Thus, the present disclosure relates generally to the spatial taggingand analysis of nucleic acids. In some instances, provided herein aremethods that utilize a transposome to fragment genomic DNA and tocapture the fragmented DNA on a spatial array, thus revealing epigenomicinsights regarding the structural features contributing to cellularregulation within the spatial context of a biological sample.

Provided herein are methods for determining location of accessiblegenomic DNA in a biological sample, the method comprising: (a) providingthe biological sample on an array comprising a plurality of captureprobes, wherein a capture probe of the plurality of capture probescomprises: (i) a spatial barcode and (ii) a capture domain; (b) addingan antibody specific to a chromatin protein in the biological sample andbinding the antibody to the chromatin protein; (c) binding atransposome-binding moiety complex to the antibody, wherein thetransposome-antibody-binding moiety complex comprises: (i) atransposase, (ii) an antibody-binding moiety, (iii) a first transposonend sequence comprising a splint sequence that is substantiallycomplementary to a portion of a splint oligonucleotide, and (iv) asecond transposon end sequence comprising a functional sequence; and (d)generating fragmented genomic DNA; (e) adding a plurality of splintoligonucleotides to the biological sample, wherein a portion of a splintoligonucleotide hybridizes to a portion of the capture domain; (f)hybridizing the splint sequence of the fragmented genomic DNA to thesplint oligonucleotide hybridized to the capture domain; and (g)determining (i) the spatial barcode or a complement thereof, of thecapture probe and (ii) all or part of a sequence of the fragmentedgenomic DNA, or a complement thereof, to determine the location of theaccessible genomic DNA in the biological sample.

In some embodiments, steps (b) and (c) are performed at the same time,and wherein the antibody and the transposome-antibody-binding moiety arecombined to form a multi-complex.

In some embodiments, the splint oligonucleotide comprises about 12 toabout 40 nucleotides.

In some embodiments, step (b), through step (f) are performed atsubstantially the same time.

In some embodiments, the methods described herein further compriseligating the splint sequence of the fragmented genomic DNA to thecapture domain of the capture probe.

In some embodiments, the methods described herein further comprise gapfilling and ligation between the 3′ end of the transposon and the 5′ endof the fragmented genomic DNA.

In some embodiments, the methods described herein further compriseextending the 3′ end of the captured fragmented genomic DNA using thecapture probe as a template, wherein the gap filling, ligation, andextension occur at the substantially the same time.

In some embodiments, the functional sequence of the second transposonend sequence comprises a primer sequence.

In some embodiments, the antibody-binding moiety is protein A, proteinG, or functional derivatives thereof.

In some embodiments, the transposase is a Tn5 transposase enzyme, a Mutransposase enzyme, a Tn7 transposase enzyme, a Vibhar speciestransposase, or functional derivatives thereof.

In some embodiments, the methods described herein further compriseextending a 3′ end of the capture probe using the fragmented genomic DNAas a template, wherein the extending step is performed using a DNApolymerase having strand displacement activity.

In some embodiments, the methods described herein further comprisestaining the biological sample, optionally wherein the stainingcomprises hematoxylin and eosin (H&E) staining or immunofluorescencestaining.

In some embodiments, the biological sample is permeabilized prior tostep (b), wherein permeabilization is chemical or enzymatic, and whereinthe chemical permeabilization condition comprises a detergent,optionally wherein the detergent is one or more of NP-40,polysorbate-20, and digitonin.

In some embodiments, the enzymatic permeabilization condition comprisesa protease of the group consisting of a pepsin, a collagenase, aproteinase K, or combinations thereof.

In some embodiments, the biological sample is a fresh tissue sample orsection, a frozen tissue sample or section, or a fixed tissue sample orsection,

In some embodiments, the fixed tissue sample or section is aformalin-fixed, paraffin embedded (FFPE) tissue sample or section.

In some embodiments, the capture probe further comprises a cleavagedomain, one or more functional domains, a unique molecular identifier,or combinations thereof.

In some embodiments, the methods described herein further comprisedetermining the location of an mRNA in the biological sample, the methodcomprising: hybridizing the mRNA or a portion thereof to the capturedomain; and determining (i) the spatial barcode or a complement thereof,and (ii) all or part of a sequence of the mRNA, or a complement thereof,and using the determined sequences of (i) and (ii) to determine thelocation of the mRNA in the biological sample.

In some embodiments, hybridizing the mRNA or a portion thereof to thecapture domain is performed concurrent with or after step (b).

In some embodiments, determining (i) the spatial barcode or a complementthereof, and (ii) all or part of a sequence of the mRNA, or a complementthereof occurs concurrent with step (g).

In some embodiments, determining (i) spatial barcode or a complementthereof, and (ii) all or part of a sequence of the mRNA, or a complementthereof comprises sequencing.

Also provided herein are kits for determining the location of accessiblegenomic DNA in a biological sample comprising: (a) an array comprising aplurality of capture probes, wherein a capture probe of the plurality ofcapture probes comprises: (i) a spatial barcode and (ii) a capturedomain; (b) a complex comprising: (i) an antibody-binding protein, (ii)a transposase, (iii) a first transposon end sequence comprising a splintsequence that is substantially complementary to a portion of a splintoligonucleotide, and (iv) a second transposon end sequence comprising afunctional sequence; and (c) instructions for performing any one of themethods described herein.

Also provided herein are kits for determining abundance and location ofaccessible genomic DNA in a biological sample comprising: (a) an arraycomprising a plurality of capture probes, wherein a capture probe of theplurality of capture probes comprises: (i) a spatial barcode and (ii) acapture domain; (b) a multi-complex comprising: (i) an antibody-bindingprotein, (ii) a transposase, (iii) a first transposon end sequencecomprising a splint sequence that is substantially complementary to aportion of a splint oligonucleotide, (iv) a second transposon endsequence comprising a functional sequence, and (v) an antibody thatbinds to a chromatin protein in the biological sample; and (c)instructions for performing any one of the methods described herein.

Also provided herein are compositions for determining abundance and/orlocation of accessible genomic DNA in a biological sample comprising:(a) an array comprising a plurality of capture probes, wherein a captureprobe of the plurality of capture probes comprises: (i) a spatialbarcode and (ii) a capture domain; (b) a complex comprising: (i) anantibody-binding protein, (ii) a transposase, (iii) a first transposonend sequence comprising a splint sequence that is substantiallycomplementary to a portion of a splint oligonucleotide, and (iv) asecond transposon end sequence; and (c) an antibody bound to a chromatinprotein in the biological sample, wherein the antibody is additionallybound to the complex from step (b).

Also provided herein are methods for determining the location ofaccessible genomic DNA in a biological sample, the method comprising:(a) providing the biological sample on a first substrate; (b) binding anantibody specific to a chromatin protein in the biological sample to thechromatin protein; (c) binding a transposome-binding moiety complex tothe antibody, wherein the transposome-antibody-binding moiety complexcomprises: (i) a transposase, (ii) an antibody-binding moiety, (iii) afirst transposon end sequence comprising a splint sequence that issubstantially complementary to a portion of a splint oligonucleotide,and (iv) a second transposon end sequence comprising a functionalsequence; and (d) generating fragmented genomic DNA; (e) aligning thefirst substrate with a second substrate comprising an array, such thatat least a portion of the biological sample is aligned with at least aportion of the array, wherein the array comprises a plurality of captureprobes, wherein a capture probe of the plurality of capture probescomprises: (i) a spatial barcode and (ii) a capture domain; (f) adding aplurality of splint oligonucleotides to the biological sample, wherein aportion of a splint oligonucleotide hybridizes to a portion of thecapture domain; (g) hybridizing the splint sequence of the fragmentedgenomic DNA to the splint oligonucleotide hybridized to the capturedomain; and (h) determining (i) the spatial barcode or a complementthereof, of the capture probe and (ii) all or part of a sequence of thefragmented genomic DNA, or a complement thereof, to determine thelocation of the accessible genomic DNA in the biological sample.

In some embodiments, the aligning comprises: mounting the firstsubstrate on a first member of a support device, the first memberconfigured to retain the first substrate; mounting the second substrateon a second member of the support device; applying a reagent medium tothe first substrate and/or the second substrate; and operating analignment mechanism of the support device to move the first memberand/or the second member such that at least a portion of the biologicalsample is aligned with at least a portion of the array, and such thatthe portion of the biological sample and the portion of the arraycontact the reagent medium.

In some embodiments, the reagent medium comprises a permeabilizationagent selected from trypsin, pepsin, elastase, or proteinase K.

In some embodiments, at least one of the first substrate and the secondsubstrate further comprise a spacer disposed on the first substrate orthe second substrate, wherein when at least the portion of thebiological sample is aligned with at least a portion of the array suchthat the portion of the biological sample and the portion of the arraycontact the reagent medium, the spacer is disposed between the firstsubstrate and the second substrate and is configured to maintain thereagent medium within a chamber formed by the first substrate, thesecond substrate, and the spacer, and to maintain a separation distancebetween the first substrate and the second substrate, wherein the spaceris positioned to surround an area on the first substrate on which thebiological sample is disposed and/or the array disposed on the secondsubstrate, wherein the area of the first substrate, the spacer, and thesecond substrate at least partially encloses a volume comprising thebiological sample.

In some embodiments, steps (b) and (c) are performed at the same time,and the antibody and the transposome-antibody-binding moiety arecombined to form a multi-complex.

In some embodiments, the transposase is a Tn5 transposase enzyme, a Mutransposase enzyme, a Tn7 transposase enzyme, a Vibhar speciestransposase, or functional derivatives thereof.

Also provided herein are methods for determining abundance and/orlocation of accessible genomic DNA in a biological sample, the methodcomprising (a) providing the biological sample on an array comprising aplurality of capture probes, wherein a capture probe of the plurality ofcapture probes comprises: (i) a spatial barcode and (ii) a capturedomain; (b) adding an antibody that binds to a chromatin protein; (c)binding a transposome-antibody-binding moiety complex to the antibody,thereby generating fragmented genomic DNA, wherein thetransposome-antibody-binding moiety complex comprises: (i) atransposase, (ii) an antibody-binding moiety (iii) a first transposonend sequence comprising a splint sequence that is substantiallycomplementary to a portion of a splint oligonucleotide, and (iv) asecond transposon end sequence comprising a functional sequence; and (d)adding a plurality of splint oligonucleotides to the biological sample,wherein a portion of a splint oligonucleotide of the plurality of splintoligonucleotides hybridizes to a portion of the capture domain; (e)hybridizing the splint sequence of the fragmented genomic DNA to thesplint oligonucleotide and hybridizing the splint oligonucleotide to thecapture probe; (f) ligating the splint sequence of the fragmentedgenomic DNA to the capture domain; and (g) determining (i) all or partof a sequence of the spatial barcode or a complement thereof, and (ii)all or part of a sequence of the fragmented genomic DNA, or a complementthereof, and using the determined sequences of (i) and (ii) to determinethe abundance and/or location of the accessible genomic DNA in thebiological sample.

Also provided herein are methods for determining abundance and/orlocation of accessible genomic DNA in a biological sample, the methodcomprising: (a) providing the biological sample on an array comprising aplurality of capture probes, wherein a capture probe of the plurality ofcapture probes comprises: (i) a spatial barcode and (ii) a capturedomain; (b) complexing an antibody that binds to a chromatin protein inthe biological sample to a transposome-antibody-binding moiety complex,generating a multi-complex, wherein the multi-complex comprises: (i) atransposase, (ii) an antibody-binding moiety, (iii) the first transposonend sequence comprising a splint sequence that is substantiallycomplementary to a portion of a splint oligonucleotide, (iv) the secondtransposon end sequence comprising a functional sequence, and (v) theantibody; and (c) adding the multi-complex to the biological sample,thereby generating fragmented genomic DNA; (d) adding a plurality ofsplint oligonucleotides to the biological sample, wherein a portion of asplint oligonucleotide of the plurality of splint oligonucleotideshybridizes to a portion of the capture domain; (e) hybridizing thesplint sequence of the fragmented genomic DNA to the splintoligonucleotide and hybridizing the splint oligonucleotide to thecapture probe, (f) ligating the transposon splint sequence of thefragmented genomic DNA to the capture domain; and (g) determining (i)all or part of a sequence of the spatial barcode or a complementthereof, and (ii) all or part of a sequence of the fragmented genomicDNA, or a complement thereof, and using the determined sequences of (i)and (ii) to determine the abundance and/or location of the accessiblegenomic DNA in the biological sample.

In some instances, the splint oligonucleotide comprises about 12 toabout 40 nucleotides.

In some instances, step (b), step (c), step (d), and step (e) areperformed at substantially the same time.

In some instances, the ligating utilizes a DNA ligase.

In some instances, any of the methods described herein further compriseperforming gap filling and ligation between the 3′ end of a transposonand a 5′ end of a fragmented genomic DNA. In some instances, any of themethods described herein further comprise extending the 3′ end of thecaptured fragmented genomic DNA using the capture probe as a template.

In some instances, the gap filling, ligation, and extension occur at thesame time.

In some instances, the functional sequence of the second transposon endsequence is a primer sequence.

In some instances, the antibody-binding moiety is protein A orfunctional derivatives thereof. In some instances, the antibody-bindingmoiety is protein G or functional derivatives thereof. In someinstances, the transposase is a Tn5 transposase enzyme, a Mu transposaseenzyme, a Tn7 transposase enzyme, a Vibhar species transposase, orfunctional derivatives thereof.

In some instances, any of the methods described herein further compriseextending a 3′ end of the capture probe using the fragmented genomic DNAas a template. In some instances, the extending step is performed usinga DNA polymerase having strand displacement activity. In some instances,the determining step (g) comprises sequencing (i) all or part of thesequence of the spatial barcode or a complement thereof, and (ii) all orpart of the sequence of the fragmented genomic DNA or a complementthereof.

In some instance, any of the methods herein further comprise stainingthe biological sample, optionally with hematoxylin and eosin (H&E)staining or immunofluorescence staining.

In some instances, adding the antibody or the multi-complex to thebiological sample is performed under a chemical permeabilizationcondition, under an enzymatic permeabilization condition, or both. Insome instances, the chemical permeabilization condition comprises adetergent. In some instances, the detergent is one or more of NP-40,polysorbate-20, and digitonin. In some instances, adding the antibody orthe multi-complex to the biological sample is performed after anenzymatic pre-permeabilization condition. In some instances, theenzymatic pre-permeabilization condition comprises a protease. In someinstances, the protease is a pepsin, a collagenase, a proteinase K, andcombinations thereof.

In some instances, the biological sample is a fresh tissue sample, afrozen tissue sample, or a fixed tissue sample. In some instances, thebiological sample is a formalin-fixed, paraffin embedded (FFPE) tissuesample. In some instances, the biological sample is a tissue section.

In some instances, the array comprises one or more features. In someinstances, the capture probe further comprises a cleavage domain, one ormore functional domains, a unique molecular identifier, or combinationsthereof.

In some instances, any of the methods herein further comprisedetermining abundance and location of an analyte in the biologicalsample, the method comprising: hybridizing the analyte or a portionthereof to the capture domain; and determining (i) all or part of asequence of the spatial barcode or a complement thereof, and (ii) all orpart of a sequence of the analyte, or a complement thereof, and usingthe determined sequences of (i) and (ii) to determine the abundance andthe location of the analyte in the biological sample.

In some instances, the analyte is RNA. In some instances, the RNA ismRNA.

In some instances, hybridizing the analyte or a portion thereof to thecapture domain is performed at the same time as step (e).

In some instances, determining (i) all or part of a sequence of thespatial barcode or a complement thereof, and (ii) all or part of asequence of the analyte, or a complement thereof occurs at the same timeas step (g). In some instances, determining (i) all or part of asequence of the spatial barcode or a complement thereof, and (ii) all orpart of a sequence of the analyte, or a complement thereof comprisessequencing (i) all or part of the sequence of the spatial barcode or acomplement thereof, and (ii) all or part of the sequence of thefragmented genomic DNA or a complement thereof.

Also provided herein are kits for determining a location of accessiblegenomic DNA in a biological sample comprising: (a) an array comprising aplurality of capture probes, wherein a capture probe of the plurality ofcapture probes comprises: (i) a spatial barcode and (ii) a capturedomain; (b) a complex comprising: (i) an antibody-binding moiety, (ii) atransposase, (iii) a first transposon end sequence comprising a splintsequence that is substantially complementary to a portion of a splintoligonucleotide, and (iv) a second transposon end sequence comprising afunctional sequence; and (c) instructions for performing any one of themethods described herein.

Also provided herein are kits for determining abundance and/or locationof accessible genomic DNA in a biological sample comprising: (a) anarray comprising a plurality of capture probes, wherein a capture probeof the plurality of capture probes comprises: (i) a spatial barcode and(ii) a capture domain; (b) a multi-complex comprising: (i) anantibody-binding moiety, (ii) a transposase, (iii) a first transposonend sequence comprising a splint sequence that is substantiallycomplementary to a portion of a splint oligonucleotide, (iv) a secondtransposon end sequence comprising a functional sequence, and (v) anantibody that binds to a chromatin protein; and (c) instructions forperforming any one of the methods described herein.

Also provided herein are compositions for determining abundance and/orlocation of accessible genomic DNA in a biological sample comprising:(a) an array comprising a plurality of capture probes, wherein a captureprobe of the plurality of capture probes comprises: (i) a spatialbarcode and (ii) a capture domain; and (b) a complex comprising: (i) anantibody-binding moiety, (ii) a transposase, (iii) a first transposonend sequence comprising a splint sequence that is substantiallycomplementary to a portion of a splint oligonucleotide, and (iv) asecond transposon end sequence.

Also provided herein are compositions for determining abundance and/orlocation of accessible genomic DNA in a biological sample comprising:(a) an array comprising a plurality of capture probes, wherein a captureprobe of the plurality of capture probes comprises: (i) a spatialbarcode and (ii) a capture domain; and (b) a multi-complex comprising:(i) an antibody-binding moiety, (ii) a transposase, (iii) a firsttransposon end sequence comprising a splint sequence that issubstantially complementary to a portion of a splint oligonucleotide,(iv) a second transposon end sequence, and (v) an antibody that binds toa chromatin protein.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, patent application, or item ofinformation was specifically and individually indicated to beincorporated by reference. To the extent publications, patents, patentapplications, and items of information incorporated by referencecontradict the disclosure contained in the specification, thespecification is intended to supersede and/or take precedence over anysuch contradictory material.

Where values are described in terms of ranges, it should be understoodthat the description includes the disclosure of all possible sub-rangeswithin such ranges, as well as specific numerical values that fallwithin such ranges irrespective of whether a specific numerical value orspecific sub-range is expressly stated.

The term “each,” when used in reference to a collection of items, isintended to identify an individual item in the collection but does notnecessarily refer to every item in the collection, unless expresslystated otherwise, or unless the context of the usage clearly indicatesotherwise.

Various instances of the features of this disclosure are describedherein. However, it should be understood that such instances areprovided merely by way of example, and numerous variations, changes, andsubstitutions can occur to those skilled in the art without departingfrom the scope of this disclosure. It should also be understood thatvarious alternatives to the specific instances described herein are alsowithin the scope of this disclosure.

BRIEF DESCRIPTION OF DRAWINGS

The following drawings illustrate certain instances of the features andadvantages of this disclosure. These instances are not intended to limitthe scope of the appended claims in any manner. Like reference symbolsin the drawings indicate like elements.

FIG. 1A shows an exemplary sandwiching process where a first substrate(e.g., a slide), including a biological sample, and a second substrate(e.g., array slide) are brought into proximity with one another.

FIG. 1B shows a fully formed sandwich configuration creating a chamberformed from one or more spacers, the first substrate, and the secondsubstrate.

FIG. 2A shows a perspective view of an exemplary sample handlingapparatus in a closed position.

FIG. 2B shows a perspective view of an exemplary sample handlingapparatus in an open position.

FIG. 3A shows the first substrate angled over (superior to) the secondsubstrate.

FIG. 3B shows that as the first substrate lowers, and/or as the secondsubstrate rises, the dropped side of the first substrate may contact adrop of reagent medium.

FIG. 3C shows a full closure and sandwiching of the first substrate andthe second substrate with one or more spacers contacting both the firstsubstrate and the second substrate.

FIG. 4A shows a side view of the angled closure workflow.

FIG. 4B shows a top view of the angled closure workflow.

FIG. 5 is a schematic diagram showing an example of a barcoded captureprobe, as described herein.

FIG. 6 is a schematic diagram showing an exemplary workflow forRNA-templated ligation.

FIG. 7 is a schematic diagram of an exemplary analyte capture agent.

FIG. 8 is a schematic diagram depicting an exemplary interaction betweena feature-immobilized capture probe 824 and an analyte capture agent826.

FIG. 9 shows an exemplary transposome complex. For example, atransposome, depicted here as a Tn5 transposome, can be conjugated to anantibody-binding protein, such as protein A (pA) or protein G (pG),thereby creating a transposome-antibody-binding protein complex, such asa protein A/G-transposome complex. ME: mosaic ends; X1: sequencecomplimentary to a splint oligo; pA/G: protein A antibody, protein Gantibody-binding protein, or an antibody-binding protein A/protein Gfusion; R1: read 1.

FIG. 10 shows an exemplary tagmentation step using a transposome complexbound to an antibody, which is in turn bound to its antigen site on anexemplary histone.

FIG. 11 shows an exemplary spatial assay for transposase accessiblechromatin. UMI: universal molecular identifier: CS1: capture sequence 1.

DETAILED DESCRIPTION

Epigenomic methods such as CUT & Run or CUT & Tag methodologies helpidentify regulators (e.g., cis regulators and/or trans regulators) thatcontribute to dynamic cellular phenotypes. While CUT & Run and CUT & Tagmethodologies have been valuable in defining epigenetic variabilitywithin a cell population, conventional applications of these methods arelimited in their ability to spatially resolve the two- andthree-dimensional structures and associated genes that promote cellularvariation. Methods that can simultaneously assess epigenomes andlocalize gene expression in a tissue sample would be useful.

Thus, the present disclosure relates generally to the spatial taggingand analysis of nucleic acids. In some instances, provided herein aremethods that utilize a transposome to fragment genomic DNA and tocapture the fragmented DNA on a spatial array, thus revealing epigenomicinsights regarding the structural features contributing to cellularregulation within the spatial context of a biological sample.

Spatial Analysis Methods

Spatial analysis methodologies described herein can provide a vastamount of analyte and/or expression data for a variety of analyteswithin a biological sample at high spatial resolution, while retainingnative spatial context. Spatial analysis methods can include, e.g., theuse of a capture probe including a spatial barcode (e.g., a nucleic acidsequence that provides information as to the location or position of ananalyte within a cell or a tissue sample (e.g., mammalian cell or amammalian tissue sample) and a capture domain that is capable of bindingto an analyte (e.g., a protein and/or a nucleic acid) produced by and/orpresent in a cell. Spatial analysis methods and compositions can alsoinclude the use of a capture probe having a capture domain that capturesan intermediate agent for indirect detection of an analyte. For example,the intermediate agent can include a nucleic acid sequence (e.g., abarcode) associated with the intermediate agent. Detection of theintermediate agent is therefore indicative of the analyte in the cell ortissue sample.

Non-limiting aspects of spatial analysis methodologies and compositionsare described in U.S. Pat. Nos. 11,447,807, 11,352,667, 11,168,350,11,104,936, 11,008,608, 10,995,361, 10,913,975, 10,774,374, 10,724,078,10,640,816, 10,494,662, 10,480,022, 10,364,457, 10,317,321, 10,059,990,10,041,949, 10,030,261, 10,002,316, 9,879,313, 9,783,841, 9,727,810,9,593,365, 8,951,726, 8,604,182, and 7,709,198; U.S. Patent ApplicationPublication Nos. 2020/0239946, 2020/0080136, 2020/0277663, 2019/0330617,2020/0256867, 2020/0224244, 2019/0085383, and 2013/0171621; PCTPublication Nos. WO2018/091676, WO2020/176788, WO2017/144338, andWO2016/057552; Non-patent literature references Rodrigues et al.,Science 363(6434):1463-1467, 2019; Lee et al., Nat. Protoc.10(3):442-458, 2015; Trejo et al., PLoS ONE 14(2):e0212031, 2019; Chenet al., Science 348(6233):aaa6090, 2015; Gao et al., BMC Biol. 15:50,2017; and Gupta et al., Nature Biotechnol. 36:1197-1202, 2018; theVisium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev F,dated January 2022); and/or the Visium Spatial Gene Expression ReagentKits—Tissue Optimization User Guide (e.g., Rev E, dated February 2022),both of which are available at the 10× Genomics Support Documentationwebsite, and can be used herein in any combination, and each of which isincorporated herein by reference in their entireties. Furthernon-limiting aspects of spatial analysis methodologies and compositionsare described herein.

Some general terminology that may be used in this disclosure can befound in Section (I)(b) of PCT Publication No. WO2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663. Typically, a “barcode”is a label, or identifier, that conveys or is capable of conveyinginformation (e.g., information about an analyte in a sample, a bead,and/or a capture probe). A barcode can be part of an analyte, orindependent of an analyte. A barcode can be attached to an analyte. Aparticular barcode can be unique relative to other barcodes. For thepurpose of this disclosure, an “analyte” can include any biologicalsubstance, structure, moiety, or component to be analyzed. The term“target” can similarly refer to an analyte of interest.

Analytes can be broadly classified into one of two groups: nucleic acidanalytes, and non-nucleic acid analytes. Examples of non-nucleic acidanalytes include, but are not limited to, lipids, carbohydrates,peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins,phosphoproteins, specific phosphorylated or acetylated variants ofproteins, amidation variants of proteins, hydroxylation variants ofproteins, methylation variants of proteins, ubiquitylation variants ofproteins, sulfation variants of proteins, viral proteins (e.g., viralcapsid, viral envelope, viral coat, viral accessory, viralglycoproteins, viral spike, etc.), extracellular and intracellularproteins, antibodies, and antigen binding fragments. In someembodiments, the analyte(s) can be localized to subcellular location(s),including, for example, organelles, e.g., mitochondria, Golgi apparatus,endoplasmic reticulum, chloroplasts, endocytic vesicles, exocyticvesicles, vacuoles, lysosomes, etc. In some embodiments, analyte(s) canbe peptides or proteins, including without limitation antibodies andenzymes. Additional examples of analytes can be found in Section (I)(c)of PCT Publication No. WO2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. In some embodiments, an analyte can bedetected indirectly, such as through detection of an intermediate agent,for example, a ligation product or an analyte capture agent (e.g., anoligonucleotide-conjugated antibody), such as those described herein.

A “biological sample” is typically obtained from the subject foranalysis using any of a variety of techniques including, but not limitedto, biopsy, surgery, and laser capture microscopy (LCM), and generallyincludes cells and/or other biological material from the subject. Insome instances, the biological sample is an organism (e.g., a smallorganism such as a parasite). In some embodiments, a biological samplecan be a fixed and/or stained biological sample (e.g., a fixed and/orstained tissue or sample). Additional methods of fixation andpreparation are provided in this application.

In some instances, the biological sample is fixed using PAXgene. PAXgeneis a formalin-free, non-cross-linking fixative that preserves morphologyand biomolecules. It is a mixture of different alcohols, acid, and asoluble organic compound. Ergin B. et al., J Proteome Res. 2010 Oct. 1;9(10):5188-96 appears to have first developed and described PAXgene. KapM. et al., PLoS One.; 6(11):e27704 (2011) and Mathieson W. et al., Am JClin Pathol.; 146(1):25-40 (2016) both describe and evaluate PAXgene fortissue fixation. Non-limiting examples of stains include histologicalstains (e.g., hematoxylin and/or eosin) and immunological stains (e.g.,fluorescent stains). In some embodiments, a biological sample (e.g., afixed and/or stained biological sample) can be imaged. Biologicalsamples are also described in Section (I)(d) of PCT Publication No.WO2020/176788 and/or U.S. Patent Application Publication No.2020/0277663.

The following embodiments can be used with any of the methods describedherein. In some embodiments, the biological sample is imaged. In someembodiments, the biological sample is visualized or imaged using brightfield microscopy. In some embodiments, the biological sample isvisualized or imaged using fluorescence microscopy. Additional methodsof visualization and imaging are known in the art. Non-limiting examplesof visualization and imaging include expansion microscopy, bright fieldmicroscopy, dark field microscopy, phase contrast microscopy, electronmicroscopy, fluorescence microscopy, reflection microscopy, interferencemicroscopy and confocal microscopy. In some embodiments, the sample isstained and imaged prior to adding the primer to the biological sample.

In some embodiments, the method includes staining the biological sample.In some embodiments, the staining includes the use of hematoxylin andeosin. In some embodiments, a biological sample can be stained using anynumber of biological stains, including but not limited to, acridineorange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI,eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains,iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red,osmium tetroxide, propidium iodide, rhodamine, or safranin. In someinstances, the biological sample can be stained using known stainingtechniques, including Can-Grunwald, Giemsa, hematoxylin and eosin (H&E),Jenner's, Leishman, Masson's trichrome, Papanicolaou, Romanowsky,silver, Sudan, Wright's, and/or Periodic Acid Schiff (PAS) stainingtechniques. PAS staining is typically performed after formalin oracetone fixation.

In some embodiments, the staining includes the use of a detectable labelselected from the group consisting of a radioisotope, a fluorophore, achemiluminescent compound, a bioluminescent compound, or a combinationthereof.

In some embodiments, a biological sample is permeabilized with one ormore permeabilization reagents. For example, permeabilization of abiological sample can facilitate analyte capture. Exemplarypermeabilization agents and conditions are described in Section(I)(d)(ii)(13) or the Exemplary Embodiments Section of PCT PublicationNo. WO2020/176788 and/or U.S. Patent Application Publication No.2020/0277663. Briefly, in any of the methods described herein, themethod includes a step of permeabilizing the biological sample. Forexample, the biological sample can be permeabilized to facilitatetransfer of the extension products to the capture probes on the array.In some embodiments, the permeabilizing includes the use of an organicsolvent (e.g., acetone, ethanol, and methanol), a detergent (e.g.,saponin, Triton X-100™, Tween-20™, or sodium dodecyl sulfate (SDS)), anenzyme (an endopeptidase, an exopeptidase, a protease), or combinationsthereof. In some embodiments, the permeabilizing includes the use of anendopeptidase, a protease, SDS, polyethylene glycol tert-octylphenylether, polysorbate 80, and polysorbate 20, N-lauroylsarcosine sodiumsalt solution, saponin, Triton X-100™, Tween-20™, or combinationsthereof. In some embodiments, the endopeptidase is pepsin. In someembodiments, the endopeptidase is Proteinase K. Additional methods forsample permeabilization are described, for example, in Jamur et al.,Method Mol. Biol. 588:63-66, 2010, the entire contents of which areincorporated herein by reference.

Array-based spatial analysis methods involve the transfer of one or moreanalytes from a biological sample to an array of features on asubstrate, where each feature is associated with a unique spatiallocation on the array. Subsequent analysis of the transferred analytesincludes determining the identity of the analytes and the spatiallocation of the analytes within the biological sample. The spatiallocation of an analyte within the biological sample is determined basedon the feature to which the analyte is bound (e.g., directly orindirectly) on the array, and the feature's relative spatial locationwithin the array.

A “capture probe” refers to any molecule capable of capturing (directlyor indirectly) and/or labelling an analyte (e.g., an analyte ofinterest) in a biological sample. In some embodiments, the capture probeis a nucleic acid or a polypeptide. In some embodiments, the captureprobe includes a barcode (e.g., a spatial barcode and/or a uniquemolecular identifier (UMI)) and a capture domain). In some instances,the capture probe includes a homopolymer sequence, such as a poly(T)sequence. In some embodiments, a capture probe can include a cleavagedomain and/or a functional domain (e.g., a primer-binding site, such asfor next-generation sequencing (NGS)). See, e.g., Section (II)(b) (e.g.,subsections (i)-(vi)) of PCT Publication No. WO2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663. Generation of captureprobes can be achieved by any appropriate method, including thosedescribed in Section (II)(d)(ii) of PCT Publication No. WO2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

In some embodiments, the biological sample is mounted on a firstsubstrate and the substrate comprising the array of capture probes is asecond substrate. During this process, one or more analytes or analytederivatives (e.g., intermediate agents; e.g., ligation products) arereleased from the biological sample and migrate to the second substratecomprising an array of capture probes. In some embodiments, the releaseand migration of the analytes or analyte derivatives to the secondsubstrate comprising the array of capture probes occurs in a manner thatpreserves the original spatial context of the analytes in the biologicalsample. This method can be referred to as a sandwiching process, whichis described e.g., in U.S. Patent Application Pub. No. 2021/0189475 andPCT Pub. Nos. WO 2021/252747 A1, WO 2022/061152 A2, and WO 2022/140028A1.

FIG. 1A shows an exemplary sandwiching process 100 where a firstsubstrate (e.g., slide 103), including a biological sample 102 (e.g., aparasitic organism), and a second substrate (e.g., array slide 104including an array having spatially barcoded capture probes 106) arebrought into proximity with one another. As shown in FIG. 1A a liquidreagent drop (e.g., permeabilization solution 105) is introduced on thesecond substrate in proximity to the capture probes 106 and in betweenthe biological sample 102 and the second substrate (e.g., slide 104including an array having spatially barcoded capture probes 106). Thepermeabilization solution 105 may release analytes or analytederivatives (e.g., intermediate agents; e.g., ligation products) thatcan be captured by the capture probes of the array 106.

During the exemplary sandwiching process, the first substrate is alignedwith the second substrate, such that at least a portion of thebiological sample is aligned with at least a portion of the captureprobes (e.g., aligned in a sandwich configuration). As shown, the secondsubstrate (e.g., array slide 104) is in a superior position to the firstsubstrate (e.g., slide 103). In some embodiments, the first substrate(e.g., slide 103) may be positioned superior to the second substrate(e.g., slide 104). A reagent medium 105 within a gap between the firstsubstrate (e.g., slide 103) and the second substrate (e.g., slide 104)creates a liquid interface between the two substrates. The reagentmedium may be a permeabilization solution which permeabilizes and/ordigests the biological sample 102. In some embodiments wherein thebiological sample 102 has been pre-permeabilized, the reagent medium isnot a permeabilization solution. In some embodiments, analytes (e.g.,mRNA transcripts) and/or analyte derivatives (e.g., intermediate agents;e.g., ligation products) of the biological sample 102 may release fromthe biological sample, actively or passively migrate (e.g., diffuse)across the gap toward the capture probes on the array 106.Alternatively, in certain embodiments, migration of the analyte oranalyte derivative (e.g., intermediate agent; e.g., ligation product)from the biological sample is performed actively (e.g., electrophoretic,by applying an electric field to promote migration). Exemplary methodsof electrophoretic migration are described in WO 2020/176788, and US.Patent Application Pub. No. 2021/0189475, each of which is herebyincorporated by reference.

As further shown, one or more spacers 110 may be positioned between thefirst substrate (e.g., slide 103) and the second substrate (e.g., arrayslide 104 including spatially barcoded capture probes 106). The one ormore spacers 110 may be configured to maintain a separation distancebetween the first substrate and the second substrate. While the one ormore spacers 110 is shown as disposed on the second substrate, thespacer may additionally or alternatively be disposed on the firstsubstrate.

In some embodiments, the one or more spacers 110 is configured tomaintain a separation distance between first and second substrates thatis between about 2 microns and 1 mm (e.g., between about 2 microns and800 microns, between about 2 microns and 700 microns, between about 2microns and 600 microns, between about 2 microns and 500 microns,between about 2 microns and 400 microns, between about 2 microns and 300microns, between about 2 microns and 200 microns, between about 2microns and 100 microns, between about 2 microns and 25 microns, orbetween about 2 microns and 10 microns), measured in a directionorthogonal to the surface of first substrate that supports thebiological sample. In some instances, the separation distance is about2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 microns. In some embodiments, the separation distanceis less than 50 microns. In some embodiments, the separation distance isless than 25 microns. In some embodiments, the separation distance isless than 20 microns. The separation distance may include a distance ofat least 2 μm.

FIG. 1B shows a fully formed sandwich configuration 125 creating achamber 150 formed from the one or more spacers 110, the first substrate(e.g., the slide 103), and the second substrate (e.g., the slide 104including an array 106 having spatially barcoded capture probes) inaccordance with some example implementations. In the example of FIG. 1B,the liquid reagent (e.g., the permeabilization solution 105) fills thevolume of the chamber 150 and may create a permeabilization buffer thatallows analytes (e.g., mRNA transcripts and/or other molecules) oranalyte derivatives (e.g., intermediate agents; e.g., ligation products)to diffuse from the biological sample 102 toward the capture probes ofthe second substrate (e.g., slide 104). In some aspects, flow of thepermeabilization buffer may deflect transcripts and/or molecules fromthe biological sample 102 and may affect diffusive transfer of analytesor analyte derivatives (e.g., intermediate agents; e.g., ligationproducts) for spatial analysis. A partially or fully sealed chamber 150resulting from the one or more spacers 110, the first substrate, and thesecond substrate may reduce or prevent flow from undesirable convectivemovement of transcripts and/or molecules over the diffusive transferfrom the biological sample 102 to the capture probes.

The sandwiching process methods described above can be implemented usinga variety of hardware components. For example, the sandwiching processmethods can be implemented using a sample holder (also referred toherein as a support device, a sample handling apparatus, and an arrayalignment device). Further details on support devices, sample holders,sample handling apparatuses, or systems for implementing a sandwichingprocess are described in, e.g., US. Patent Application Pub. No.2021/0189475, and PCT Publ. No. WO 2022/061152 A2, each of which areincorporated by reference in their entirety.

In some embodiments of a sample holder, the sample holder can include afirst member including a first retaining mechanism configured to retaina first substrate comprising a biological sample. The first retainingmechanism can be configured to retain the first substrate disposed in afirst plane. The sample holder can further include a second memberincluding a second retaining mechanism configured to retain a secondsubstrate disposed in a second plane. The sample holder can furtherinclude an alignment mechanism connected to one or both of the firstmember and the second member. The alignment mechanism can be configuredto align the first and second members along the first plane and/or thesecond plane such that the sample contacts at least a portion of thereagent medium when the first and second members are aligned and withina threshold distance along an axis orthogonal to the second plane. Theadjustment mechanism may be configured to move the second member alongthe axis orthogonal to the second plane and/or move the first memberalong an axis orthogonal to the first plane.

In some embodiments, the adjustment mechanism includes a linearactuator. In some embodiments, the linear actuator is configured to movethe second member along an axis orthogonal to the plane of the firstmember and/or the second member. In some embodiments, the linearactuator is configured to move the first member along an axis orthogonalto the plane of the first member and/or the second member. In someembodiments, the linear actuator is configured to move the first member,the second member, or both the first member and the second member at avelocity of at least 0.1 mm/sec. In some embodiments, the linearactuator is configured to move the first member, the second member, orboth the first member and the second member with an amount of force ofat least 0.1 lbs.

FIG. 2A is a perspective view of an example sample handling apparatus200 in a closed position in accordance with some exampleimplementations. As shown, the sample handling apparatus 200 includes afirst member 204, a second member 210, optionally an image capturedevice 220, a first substrate 206, optionally a hinge 215, andoptionally a mirror 216. The hinge 215 may be configured to allow thefirst member 204 to be positioned in an open or closed configuration byopening and/or closing the first member 204 in a clamshell manner alongthe hinge 215.

FIG. 2B is a perspective view of the example sample handling apparatus200 in an open position in accordance with some example implementations.As shown, the sample handling apparatus 200 includes one or more firstretaining mechanisms 208 configured to retain one or more firstsubstrates 206. In the example of FIG. 2B, the first member 204 isconfigured to retain two first substrates 206, however the first member204 may be configured to retain more or fewer first substrates 206.

In some aspects, when the sample handling apparatus 200 is in an openposition (e.g., in FIG. 2B), the first substrate 206 and/or the secondsubstrate 212 may be loaded and positioned within the sample handlingapparatus 200 such as within the first member 204 and the second member210, respectively. As noted, the hinge 215 may allow the first member204 to close over the second member 210 and form a sandwichconfiguration.

In some aspects, after the first member 204 closes over the secondmember 210, an adjustment mechanism of the sample handling apparatus 200may actuate the first member 204 and/or the second member 210 to formthe sandwich configuration for the permeabilization step (e.g., bringingthe first substrate 206 and the second substrate 212 closer to eachother and within a threshold distance for the sandwich configuration).The adjustment mechanism may be configured to control a speed, an angle,a force, or the like of the sandwich configuration.

In some embodiments, the biological sample (e.g., sample 102 from FIG.1A) may be aligned within the first member 204 (e.g., via the firstretaining mechanism 208) prior to closing the first member 204 such thata desired region of interest of the sample is aligned with the barcodedarray of the second substrate (e.g., the slide 104 from FIG. 1A), e.g.,when the first and second substrates are aligned in the sandwichconfiguration. Such alignment may be accomplished manually (e.g., by auser) or automatically (e.g., via an automated alignment mechanism).After or before alignment, spacers may be applied to the first substrate206 and/or the second substrate 212 to maintain a minimum spacingbetween the first substrate 206 and the second substrate 212 duringsandwiching. In some aspects, the permeabilization solution (e.g.,permeabilization solution 305) may be applied to the first substrate 206and/or the second substrate 212. The first member 204 may then closeover the second member 210 and form the sandwich configuration. Analytesor analyte derivatives (e.g., intermediate agents; e.g., ligationproducts) may be captured by the capture probes of the array and may beprocessed for spatial analysis.

In some embodiments, during the permeabilization step, the image capturedevice 220 may capture images of the overlap area between the biologicalsample and the capture probes on the array 106. If more than one firstsubstrates 206 and/or second substrates 212 are present within thesample handling apparatus 200, the image capture device 220 may beconfigured to capture one or more images of one or more overlap areas.

Provided herein are methods for delivering a fluid to a biologicalsample disposed on an area of a first substrate and an array disposed ona second substrate. FIGS. 3A-3C depict a side view and a top view of anexemplary angled closure workflow 300 for sandwiching a first substrate(e.g., slide 303) having a biological sample 302 and a second substrate(e.g., slide 304 having capture probes 306) in accordance with someexemplary implementations.

FIG. 3A depicts the first substrate (e.g., the slide 303 including abiological sample 302) angled over (superior to) the second substrate(e.g., slide 304). As shown, reagent medium (e.g., permeabilizationsolution) 305 is located on the spacer 310 toward the right-hand side ofthe side view in FIG. 3A. While FIG. 3A depicts the reagent medium onthe right hand side of side view, it should be understood that suchdepiction is not meant to be limiting as to the location of the reagentmedium on the spacer.

FIG. 3B shows that as the first substrate lowers, and/or as the secondsubstrate rises, the dropped side of the first substrate (e.g., a sideof the slide 303 angled toward the second substrate) may contact thereagent medium 305. The dropped side of the first substrate may urge thereagent medium 305 toward the opposite direction (e.g., towards anopposite side of the spacer 310, towards an opposite side of the firstsubstrate relative to the dropped side). For example, in the side viewof FIG. 3B the reagent medium 305 may be urged from right to left as thesandwich is formed.

In some embodiments, the first substrate and/or the second substrate arefurther moved to achieve an approximately parallel arrangement of thefirst substrate and the second substrate.

FIG. 3C depicts a full closure of the sandwich between the firstsubstrate and the second substrate with the spacer 310 contacting boththe first substrate and the second substrate and maintaining aseparation distance and optionally the approximately parallelarrangement between the two substrates. As shown in the top view of FIG.3C, the spacer 310 fully encloses and surrounds the biological sample302 and the capture probes 306, and the spacer 310 form the sides ofchamber 350 which holds a volume of the reagent medium 305.

While FIG. 3C depicts the first substrate (e.g., the slide 303 includingbiological sample 302) angled over (superior to) the second substrate(e.g., slide 304) and the second substrate comprising the spacer 310, itshould be understood that an exemplary angled closure workflow caninclude the second substrate angled over (superior to) the firstsubstrate and the first substrate comprising the spacer 310.

It may be desirable that the reagent medium be free from air bubblesbetween the substrates to facilitate transfer of target analytes withspatial information. Additionally, air bubbles present between thesubstrates may obscure at least a portion of an image capture of adesired region of interest. Accordingly, it may be desirable to ensureor encourage suppression and/or elimination of air bubbles between thetwo substrates (e.g., slide 303 and slide 304) during a permeabilizationstep (e.g., step 104). In some aspects, it may be possible to reduce oreliminate bubble formation between the substrates using a variety offilling methods and/or closing methods. In some instances, the firstsubstrate and the second substrate are arranged in an angled sandwichassembly as described herein. For example, during the sandwiching of thetwo substrates (e.g., the slide 303 and the slide 304), an angledclosure workflow may be used to suppress or eliminate bubble formation.

FIG. 4A is a side view of the angled closure workflow 400 in accordancewith some exemplary implementations. FIG. 4B is a top view of the angledclosure workflow 400 in accordance with some exemplary implementations.As shown at 405, reagent medium 401 is positioned to the side of thesubstrate 402 contacting the spring.

At step 410, the dropped side of the angled substrate 406 contacts thereagent medium 401 first. The contact of the substrate 406 with thereagent medium 401 may form a linear or low curvature flow front thatfills uniformly with the slides closed.

At step 415, the substrate 406 is further lowered toward the substrate402 (or the substrate 402 is raised up toward the substrate 406) and thedropped side of the substrate 406 may contact and may urge the liquidreagent toward the side opposite the dropped side and creating a linearor low curvature flow front that may prevent or reduce bubble trappingbetween the substrates. As further shown, the spring may begin tocompress as the substrate 406 is lowered.

At step 420, the reagent medium 401 fills the gap between the substrate406 and the substrate 402. The linear flow front of the liquid reagentmay form by squeezing the 401 volume along the contact side of thesubstrate 402 and/or the substrate 406. Additionally, capillary flow mayalso contribute to filling the gap area. As further shown in step 420,the spring may be fully compressed such that the substrate 406, thesubstrate 402, and the base are substantially parallel to each other.

In some embodiments, the reagent medium (e.g., 105 in FIG. 1A) comprisesa permeabilization agent. In some embodiments, following initial contactbetween the biological sample and a permeabilization agent, thepermeabilization agent can be removed from contact with the biologicalsample (e.g., by opening sample holder). Suitable agents for thispurpose include, but are not limited to, organic solvents (e.g.,acetone, ethanol, and methanol), cross-linking agents (e.g.,paraformaldehyde), detergents (e.g., saponin, Triton X-100™, Tween-20™,or sodium dodecyl sulfate (SDS)), and enzymes (e.g., trypsin, proteases(e.g., proteinase K). In some embodiments, the detergent is an anionicdetergent (e.g., SDS or N-lauroylsarcosine sodium salt solution).

In some embodiments, the reagent medium comprises a lysis reagent. Lysissolutions can include ionic surfactants such as, for example, sarkosyland sodium dodecyl sulfate (SDS). More generally, chemical lysis agentscan include, without limitation, organic solvents, chelating agents,detergents, surfactants, and chaotropic agents. In some embodiments, thereagent medium comprises a protease. Exemplary proteases include, e.g.,pepsin, trypsin, pepsin, elastase, and proteinase K. In someembodiments, the reagent medium comprises a nuclease. In someembodiments, the nuclease comprises an RNase. In some embodiments, theRnase is selected from Rnase A, Rnase C, Rnase H, and Rnase I. In someembodiments, the reagent medium comprises one or more of sodium dodecylsulfate (SDS), proteinase K, pepsin, N-lauroylsarcosine, RNAse, and asodium salt thereof.

In some embodiments, the reagent medium comprises polyethylene glycol(PEG). In some embodiments, the PEG is from about PEG 2K to about PEG16K. In some embodiments, the PEG is PEG 2K, 3K, 4K, 5K, 6K, 7K, 8K, 9K,10K, 11K, 12K, 13K, 14K, 15K, or 16K. In some embodiments, the PEG ispresent at a concentration from about 2% to 25%, from about 4% to about23%, from about 6% to about 21%, or from about 8% to about 20% (v/v).

In certain embodiments a dried permeabilization reagent is applied orformed as a layer on the first substrate or the second substrate or bothprior to contacting the biological sample and the array. For example, apermeabilization reagent can be deposited in solution on the firstsubstrate or the second substrate or both and then dried.

In some instances, the aligned portions of the biological sample and thearray are in contact with the reagent medium for about 1 minute, about 5minutes, about 10 minutes, about 12 minutes, about 15 minutes, about 18minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 36minutes, about 45 minutes, or about an hour. In some instances, thealigned portions of the biological sample and the array are in contactwith the reagent medium for about 1-60 minutes.

In some instances, the device is configured to control a temperature ofthe first and second substrates. In some embodiments, the temperature ofthe first and second members is lowered to a first temperature that isbelow room temperature.

There are at least two methods to associate a spatial barcode with oneor more neighboring cells, such that the spatial barcode identifies theone or more cells, and/or contents of the one or more cells, asassociated with a particular spatial location. One method is to promoteanalytes or analyte proxies (e.g., intermediate agents) out of a celland towards a spatially-barcoded array (e.g., includingspatially-barcoded capture probes). Another method is to cleavespatially-barcoded capture probes from an array and promote thespatially-barcoded capture probes towards and/or into or onto thebiological sample.

In some cases, capture probes may be configured to prime, replicate, andconsequently yield optionally barcoded extension products from atemplate (e.g., a DNA or RNA template, such as an analyte or anintermediate agent (e.g., a ligation product or an analyte captureagent), or a portion thereof), or derivatives thereof (see, e.g.,Section (II)(b)(vii) of PCT Publication No. WO2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663 regarding extendedcapture probes). In some cases, capture probes may be configured to formligation products with a template (e.g., a DNA or RNA template, such asan analyte or an intermediate agent, or portion thereof), therebycreating ligations products that serve as proxies for the template.

As used herein, an “extended capture probe” refers to a capture probehaving additional nucleotides added to the terminus (e.g., 3′ or 5′ end)of the capture probe thereby extending the overall length of the captureprobe. For example, an “extended 3′ end” indicates additionalnucleotides were added to the most 3′ nucleotide of the capture probe toextend the length of the capture probe, for example, by polymerizationreactions used to extend nucleic acid molecules including templatedpolymerization catalyzed by a polymerase (e.g., a DNA polymerase or areverse transcriptase). In some embodiments, extending the capture probeincludes adding to a 3′ end of a capture probe a nucleic acid sequencethat is complementary to a nucleic acid sequence of an analyte orintermediate agent specifically bound to the capture domain of thecapture probe. In some embodiments, the capture probe is extended by areverse transcriptase. In some embodiments, the capture probe isextended using one or more DNA polymerases. In some embodiments, theextended capture probes include the sequence of the capture domain andthe sequence of the spatial barcode of the capture probe.

In some embodiments, extended capture probes are amplified (e.g., inbulk solution or on the array) to yield quantities that are sufficientfor downstream analysis, e.g., sequencing. In some embodiments, extendedcapture probes (e.g., DNA molecules) can act as templates for anamplification reaction (e.g., a polymerase chain reaction).

Additional variants of spatial analysis methods, including in someembodiments, an imaging step, are described in Section (II)(a) of PCTPublication No. WO2020/176788 and/or U.S. Patent Application PublicationNo. 2020/0277663. Analysis of captured analytes (and/or intermediateagents or portions thereof), for example, including sample removal,extension of capture probes, sequencing (e.g., of a cleaved extendedcapture probe and/or a cDNA molecule complementary to an extendedcapture probe), sequencing on the array (e.g., using, for example, insitu hybridization or in situ ligation approaches), temporal analysis,and/or proximity capture, is described in Section (II)(g) of PCTPublication No. WO2020/176788 and/or U.S. Patent Application PublicationNo. 2020/0277663. Some quality control measures are described in Section(II)(h) of PCT Publication No. WO2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Spatial information can provide information of medical importance. Forexample, the methods described herein can allow for: identification ofone or more biomarkers (e.g., diagnostic, prognostic, and/or fordetermination of efficacy of a treatment) of a disease or disorder;identification of a candidate drug target for treatment of a disease ordisorder; identification (e.g., diagnosis) of a subject as having adisease or disorder; identification of stage and/or prognosis of adisease or disorder in a subject; identification of a subject as havingan increased likelihood of developing a disease or disorder; monitoringof progression of a disease or disorder in a subject; determination ofefficacy of a treatment of a disease or disorder in a subject;identification of a patient subpopulation for which a treatment iseffective for a disease or disorder; modification of a treatment of asubject with a disease or disorder; selection of a subject forparticipation in a clinical trial; and/or selection of a treatment for asubject with a disease or disorder. Exemplary methods for identifyingspatial information of biological and/or medical importance can be foundin U.S. Patent Application Publication Nos. 2021/0140982, 2021/0198741,and 2021/0199660.

Spatial information can provide information of biological importance.For example, the methods described herein can allow for: identificationof transcriptome and/or proteome expression profiles (e.g., in healthyand/or diseased tissue); identification of multiple analyte types inclose proximity (e.g., nearest neighbor or proximity based analysis);determination of up- and/or down-regulated genes and/or proteins indiseased tissue; characterization of tumor microenvironments;characterization of tumor immune responses; characterization of cellstypes and their co-localization in healthy and diseased tissue; andidentification of genetic variants within tissues (e.g., based on geneand/or protein expression profiles associated with specific disease ordisorder biomarkers).

Typically, for spatial array-based methods, a substrate functions as asupport for direct or indirect attachment of capture probes to featuresof the array. A “feature” is an entity that acts as a support orrepository for various molecular entities used in spatial analysis. Insome embodiments, some or all of the features in an array arefunctionalized for analyte capture. Exemplary substrates are describedin Section (II)(c) of PCT Publication No. WO2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663. Exemplary features andgeometric attributes of an array can be found in Sections (II)(d)(i),(II)(d)(iii), and (II)(d)(iv) of PCT Publication No. WO2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

Generally, analytes and/or intermediate agents (or portions thereof) canbe captured when contacting a biological sample with a substrateincluding capture probes (e.g., a substrate with capture probesembedded, spotted, printed, fabricated on the substrate, or a substratewith features (e.g., beads, wells) comprising capture probes). As usedherein, “contact,” “contacted,” and/or “contacting,” a biological samplewith a substrate refers to any contact (e.g., direct or indirect) suchthat capture probes can interact (e.g., bind covalently ornon-covalently (e.g., hybridize)) with analytes from the biologicalsample. Capture can be achieved actively (e.g., using electrophoresis)or passively (e.g., using diffusion). Analyte capture is furtherdescribed in Section (II)(e) of PCT Publication No. WO2020/176788 and/orU.S. Patent Application Publication No. 2020/0277663.

FIG. 5 is a schematic diagram showing an exemplary capture probe, asdescribed herein. As shown, the capture probe 502 is optionally coupledto a feature 501 by a cleavage domain 503, such as a disulfide linker.The capture probe can include a functional sequence 504 that are usefulfor subsequent processing. The functional sequence 504 can include allor a part of sequencer specific flow cell attachment sequence (e.g., aP5 or P7 sequence), all or a part of a sequencing primer sequence,(e.g., a R1 primer binding site, a R2 primer binding site), orcombinations thereof. The capture probe can also include a spatialbarcode 505. The capture probe can also include a unique molecularidentifier (UMI) sequence 506. While FIG. 5 shows the spatial barcode505 as being located upstream (5′) of UMI sequence 506, it is to beunderstood that capture probes wherein UMI sequence 506 is locatedupstream (5′) of the spatial barcode 505 is also suitable for use in anyof the methods described herein. The capture probe can also include acapture domain 507 to facilitate capture of a target analyte. Thecapture domain can have a sequence complementary to a sequence of anucleic acid analyte. The capture domain can have a sequencecomplementary to a connected probe described herein. The capture domaincan have a sequence complementary to a capture handle sequence presentin an analyte capture agent. The capture domain can have a sequencecomplementary to a splint oligonucleotide. Such splint oligonucleotide,in addition to having a sequence complementary to a capture domain of acapture probe, can have a sequence complementary to a sequence of anucleic acid analyte, a portion of a connected probe described herein, acapture handle sequence described herein, and/or a methylated adaptordescribed herein.

The functional sequences can generally be selected for compatibilitywith any of a variety of different sequencing systems, e.g., Ion TorrentProton or PGM, Illumina sequencing instruments, PacBio, Oxford Nanopore,etc., and the requirements thereof. In some embodiments, functionalsequences can be selected for compatibility with non-commercializedsequencing systems. Examples of such sequencing systems and techniques,for which suitable functional sequences can be used, include (but arenot limited to) Ion Torrent Proton or PGM sequencing, Illuminasequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing.Further, in some embodiments, functional sequences can be selected forcompatibility with other sequencing systems, includingnon-commercialized sequencing systems.

In some embodiments, the spatial barcode 505 and functional sequences504 is common to all of the probes attached to a given feature. In someembodiments, the UMI sequence 506 of a capture probe attached to a givenfeature is different from the UMI sequence of a different capture probeattached to the given feature.

In some embodiments, more than one analyte type (e.g., nucleic acids andproteins) from a biological sample can be detected (e.g., simultaneouslyor sequentially) using any appropriate multiplexing technique, such asthose described in Section (IV) of PCT Publication No. WO2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by attaching and/orintroducing a molecule (e.g., a peptide, a lipid, or a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) to a biologicalsample (e.g., to a cell in a biological sample). In some embodiments, aplurality of molecules (e.g., a plurality of nucleic acid molecules)having a plurality of barcodes (e.g., a plurality of spatial barcodes)are introduced to a biological sample (e.g., to a plurality of cells ina biological sample) for use in spatial analysis. In some embodiments,after attaching and/or introducing a molecule having a barcode to abiological sample, the biological sample can be physically separated(e.g., dissociated) into single cells or cell groups for analysis. Somesuch methods of spatial analysis are described in Section (III) of PCTPublication No. WO2020/176788 and/or U.S. Patent Application PublicationNo. 2020/0277663.

In some cases, spatial analysis can be performed by detecting multipleoligonucleotides that hybridize to an analyte. In some instances, forexample, spatial analysis can be performed using RNA-templated ligation(RTL). Methods of RTL have been described previously. See, e.g., Credleet al., Nucleic Acids Res. 2017 Aug. 21; 45(14):e128. Typically, RTLincludes hybridization of two oligonucleotides to adjacent sequences onan analyte (e.g., an RNA molecule, such as an mRNA molecule). In someinstances, the oligonucleotides are DNA molecules. In some instances,one of the oligonucleotides includes at least two ribonucleic acid basesat the 3′ end and/or the other oligonucleotide includes a phosphorylatednucleotide at the 5′ end. In some instances, one of the twooligonucleotides includes a capture binding capture domain (e.g., apoly(A) sequence, a non-homopolymeric sequence). After hybridization tothe analyte, a ligase (e.g., a T4 RNA ligase (Rnl2), a PBCV-1 DNA Ligaseor Chorella virus DNA Ligase, a single-stranded DNA ligase, or a T4 DNAligase) ligates the two oligonucleotides together, creating a ligationproduct. In some instances, the two oligonucleotides hybridize tosequences that are not adjacent to one another. For example,hybridization of the two oligonucleotides creates a gap between thehybridized oligonucleotides. In some instances, a polymerase (e.g., aDNA polymerase) can extend one of the oligonucleotides prior toligation. After ligation, the ligation product is released from theanalyte. In some instances, the ligation product is released using anendonuclease (e.g., RNAse H). In some instances, the ligation product isremoved using heat. In some instances, the ligation product is removedusing KOH. The released ligation product can then be captured by captureprobes (e.g., instead of direct capture of an analyte) on an array,optionally amplified, and sequenced, thus determining the location andoptionally the abundance of the analyte in the biological sample.

A non-limiting example of templated ligation methods disclosed herein isdepicted in FIG. 6 . After a biological sample is contacted with asubstrate including a plurality of capture probes and contacted with (a)a first probe 601 having a target-hybridization sequence 603 and aprimer sequence 602 and (b) a second probe 604 having atarget-hybridization sequence 605 and a capture domain (e.g., a poly-Asequence) 606, the first probe 601 and a second probe 604 hybridize 610to an analyte 607. A ligase 621 ligates 620 the first probe to thesecond probe thereby generating a ligation product 622. The ligationproduct is released 630 from the analyte 631 by digesting the analyteusing an endoribonuclease 632. The sample is permeabilized 640 and theligation product 641 is able to hybridize to a capture probe on thesubstrate. Methods and composition for spatial detection using templatedligation have been described in PCT Publ. No. WO 2021/133849 A1, U.S.Pat. No. 11,332,790 and 11,505,828, each of which is incorporated byreference in its entirety.

In some embodiments, detection of one or more analytes (e.g., proteinanalytes) can be performed using one or more analyte capture agents. Asused herein, an “analyte capture agent” refers to an agent thatinteracts with an analyte (e.g., an analyte in a biological sample) andwith a capture probe (e.g., a capture probe attached to a substrate or afeature) to identify the analyte. In some embodiments, the analytecapture agent includes: (i) an analyte binding moiety (e.g., that bindsto an analyte), for example, an antibody or antigen-binding fragmentthereof; (ii) analyte binding moiety barcode; and (iii) an analytecapture sequence. As used herein, the term “analyte binding moietybarcode” refers to a barcode that is associated with or otherwiseidentifies the analyte binding moiety. As used herein, the term “analytecapture sequence” refers to a region or moiety configured to hybridizeto, bind to, couple to, or otherwise interact with a capture domain of acapture probe. In some cases, an analyte binding moiety barcode (orportion thereof) may be able to be removed (e.g., cleaved) from theanalyte capture agent. Additional description of analyte capture agentscan be found in Section (II)(b)(ix) of PCT Publication No. WO2020/176788and/or Section (II)(b)(viii) U.S. Patent Application Publication No.2020/0277663.

FIG. 7 is a schematic diagram of an exemplary analyte capture agent 702comprised of an analyte-binding moiety 704 and an analyte-binding moietybarcode domain 708. The exemplary analyte-binding moiety 704 is amolecule capable of binding to an analyte 706 and the analyte captureagent is capable of interacting with a spatially-barcoded capture probe.The analyte-binding moiety can bind to the analyte 706 with highaffinity and/or with high specificity. The analyte capture agent caninclude an analyte-binding moiety barcode domain 708, a nucleotidesequence (e.g., an oligonucleotide), which can hybridize to at least aportion or an entirety of a capture domain of a capture probe. Theanalyte-binding moiety barcode domain 708 can comprise an analytebinding moiety barcode and a capture handle sequence described herein.The analyte-binding moiety 704 can include a polypeptide and/or anaptamer. The analyte-binding moiety 704 can include an antibody orantibody fragment (e.g., an antigen-binding fragment).

FIG. 8 is a schematic diagram depicting an exemplary interaction betweena feature-immobilized capture probe 824 and an analyte capture agent826. The feature-immobilized capture probe 824 can include a spatialbarcode 808 as well as functional sequences 806 and UMI 810, asdescribed elsewhere herein. The capture probe can be affixed 804 to afeature (e.g., bead) or array 802. The capture probe can also include acapture domain 812 that is capable of binding to an analyte captureagent 826. The analyte capture agent 826 can include a functionalsequence 818, analyte binding moiety barcode 816, and a capture handlesequence 814 that is capable of binding to the capture domain 812 of thecapture probe 824. The analyte capture agent can also include a linker820 that allows the capture agent barcode domain 816 to couple to theanalyte binding moiety 822.

During analysis of spatial information, sequence information for aspatial barcode associated with an analyte is obtained, and the sequenceinformation can be used to provide information about the spatialdistribution of the analyte in the biological sample. Various methodscan be used to obtain the spatial information. In some embodiments,specific capture probes and the analytes they capture are associatedwith specific locations in an array of features on a substrate. Forexample, specific spatial barcodes can be associated with specific arraylocations prior to array fabrication, and the sequences of the spatialbarcodes can be stored (e.g., in a database) along with specific arraylocation information, so that each spatial barcode uniquely maps to aparticular array location.

Alternatively, specific spatial barcodes can be deposited atpredetermined locations in an array of features during fabrication suchthat at each location, only one type of spatial barcode is present sothat spatial barcodes are uniquely associated with a single feature ofthe array. Where necessary, the arrays can be decoded using any of themethods described herein so that spatial barcodes are uniquelyassociated with array feature locations, and this mapping can be storedas described above.

When sequence information is obtained for capture probes and/or analytesduring analysis of spatial information, the locations of the captureprobes and/or analytes can be determined by referring to the storedinformation that uniquely associates each spatial barcode with an arrayfeature location. In this manner, specific capture probes and capturedanalytes are associated with specific locations in the array offeatures. Each array feature location represents a position relative toa coordinate reference point (e.g., an array location, a fiducialmarker) for the array. Accordingly, each feature location has an“address” or location in the coordinate space of the array.

Some exemplary spatial analysis workflows are described in the ExemplaryEmbodiments section of PCT Publication No. WO2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663. See, for example, theExemplary embodiment starting with “In some non-limiting examples of theworkflows described herein, the sample can be immersed . . . ” of PCTPublication No. WO2020/176788 and/or U.S. Patent Application PublicationNo. 2020/0277663. See also, e.g., the Visium Spatial Gene ExpressionReagent Kits User Guide (e.g., Rev F, dated January 2022); and/or theVisium Spatial Gene Expression Reagent Kits—Tissue Optimization UserGuide (e.g., Rev E, dated February 2022).

In some embodiments, spatial analysis can be performed using dedicatedhardware and/or software, such as any of the systems described inSections (II)(e)(ii) and/or (V) of PCT Publication No. WO2020/176788and/or U.S. Patent Application Publication No. 2020/0277663, or any ofone or more of the devices or methods described in Sections ControlSlide for Imaging, Methods of Using Control Slides and Substrates for,Systems of Using Control Slides and Substrates for Imaging, and/orSample and Array Alignment Devices and Methods, Informational labels ofPCT Publication No. WO2020/123320.

Suitable systems for performing spatial analysis can include componentssuch as a chamber (e.g., a flow cell or sealable, fluid-tight chamber)for containing a biological sample. The biological sample can be mountedfor example, in a biological sample holder. One or more fluid chamberscan be connected to the chamber and/or the sample holder via fluidconduits, and fluids can be delivered into the chamber and/or sampleholder via fluidic pumps, vacuum sources, or other devices coupled tothe fluid conduits that create a pressure gradient to drive fluid flow.One or more valves can also be connected to fluid conduits to regulatethe flow of reagents from reservoirs to the chamber and/or sampleholder.

The systems can optionally include a control unit that includes one ormore electronic processors, an input interface, an output interface(such as a display), and a storage unit (e.g., a solid state storagemedium such as, but not limited to, a magnetic, optical, or other solidstate, persistent, writeable and/or re-writeable storage medium). Thecontrol unit can optionally be connected to one or more remote devicesvia a network. The control unit (and components thereof) can generallyperform any of the steps and functions described herein. Where thesystem is connected to a remote device, the remote device (or devices)can perform any of the steps or features described herein. The systemscan optionally include one or more detectors (e.g., CCD, CMOS) used tocapture images. The systems can also optionally include one or morelight sources (e.g., LED-based, diode-based, lasers) for illuminating asample, a substrate with features, analytes from a biological samplecaptured on a substrate, and various control and calibration media.

The systems can optionally include software instructions encoded and/orimplemented in one or more of tangible storage media and hardwarecomponents such as application specific integrated circuits. Thesoftware instructions, when executed by a control unit (and inparticular, an electronic processor) or an integrated circuit, can causethe control unit, integrated circuit, or other component executing thesoftware instructions to perform any of the method steps or functionsdescribed herein.

In some cases, the systems described herein can detect (e.g., registeran image) the biological sample on the array. Exemplary methods todetect the biological sample on an array are described in PCTPublication No. WO2021/102003 and/or U.S. Patent Application PublicationNo. 2021/0150707, each of which is incorporated herein by reference intheir entireties.

Prior to transferring analytes from the biological sample to the arrayof features on the substrate, the biological sample can be aligned withthe array. Alignment of a biological sample and an array of featuresincluding capture probes can facilitate spatial analysis, which can beused to detect differences in analyte presence and/or level withindifferent positions in the biological sample, for example, to generate athree-dimensional map of the analyte presence and/or level. Exemplarymethods to generate a two- and/or three-dimensional map of the analytepresence and/or level are described in PCT Publication No. WO2020/053655and spatial analysis methods are generally described in PCT PublicationNo. WO2021/102039 and/or U.S. Patent Application Publication No.2021/0155982, each of which is incorporated herein by reference in theirentireties.

In some cases, a map of analyte presence and/or level can be aligned toan image of a biological sample using one or more fiducial markers,e.g., objects placed in the field of view of an imaging system whichappear in the image produced, as described in the Substrate AttributesSection, Control Slide for Imaging Section of PCT Publication Nos.WO2020/123320, WO 2021/102005, and/or U.S. Patent ApplicationPublication No. 2021/0158522, each of which is incorporated herein byreference in their entireties. Fiducial markers can be used as a pointof reference or measurement scale for alignment (e.g., to align a sampleand an array, to align two substrates, to determine a location of asample or array on a substrate relative to a fiducial marker) and/or forquantitative measurements of sizes and/or distances.

Spatial Assay for Transposase Accessible Chromatin

The human body includes a large collection of diverse cell types, eachproviding a specialized and context-specific function. Understanding acell's chromatin structure can reveal information about a cell'sfunction. Open chromatin, or accessible chromatin, or accessible genomicDNA, is often indicative of transcriptionally active sequences, e.g.,genes, in a particular cell. Further understanding the transcriptionallyactive regions within chromatin will enable identification of whichgenes contribute to a cell's function and/or phenotype.

Methods have been developed to study epigenomes, e.g., chromatinaccessibility assays (ATAC-seq, CUT & RUN, Cleavage Under Targets andTagmentation (CUT & Tag)) or identifying proteins associated withchromatin e.g., (ChIP-seq). These assays help identify, for example,regulators (e.g., cis regulators and/or trans regulators) thatcontribute to dynamic cellular phenotypes. See, e.g., CUT & RUN and CUT& Tag described in Skene and Henikoff, eLife 2017; 6:e21856; Meers etal., eLIFE (2019) 8:e46314; and Kaya-Okur et al., Nature Communications(2019):10, 1930, each of which is incorporated by reference in itsentirety. See also, e.g., single-cell CUT & Tag described in Bartosovicet al., Nature Biotechnology (2021):39, 825-835; Deng et al. bioRxiv(2021) preprint doi: https://doi.org/10.1101/2021.03.11.434985, each ofwhich is incorporated by reference in its entirety. While CUT & RUN andCUT & Tag have been invaluable in defining epigenetic variability withina cell population, conventional applications of these methods arelimited in their ability to spatially resolve the three dimensionalstructures and associated genes that promote cellular variation.

Thus, the present disclosure relates generally to the spatial taggingand analysis of nucleic acids. In some instances, provided herein aremethods that utilize a transposase enzyme fused to an antibody-bindingmoiety (e.g. an antibody-binding protein that contains anantibody-binding moiety, such as protein A, protein G, fusion of proteinA and protein G, or functional equivalent thereof), to engage andfragment, for example, the accessible (e.g., open chromatin) genomic DNAand enable the simultaneous capture of DNA and RNA from a biologicalsample, thus revealing epigenomic insights regarding the structuralfeatures contributing to cellular regulation.

In some instances of any of the spatial analysis methods describedherein, CUT & Tag based methods or methods based on assays fortransposase-accessible chromatin using sequencing (ATAC-seq) is used togenerate genome-wide chromatin accessibility maps. These genome-wideaccessibility maps can be integrated with additional genome-wideprofiling data (e.g., RNA-seq, ChIP-seq, Methyl-Seq) to produce generegulatory interaction maps that facilitate understanding oftranscriptional regulation. For example, interrogation of genome-wideaccessibility maps can reveal the underlying transcription factors andthe transcription factor motifs responsible for chromatin accessibilityat a given genomic location. Correlating changes in chromatinaccessibility with changes in gene expression (RNA-seq), changes intranscription factor (TF) binding (e.g., ChIP-seq) and/or changes in DNAmethylation levels (e.g., Methyl-seq) can identify the transcriptionregulation driving these changes. In disease states, there is often animbalance in this transcriptional regulation. Thus, analyzing bothchromatin accessibility and, for example, gene expression using spatialanalysis methods enables identification of causes underlying theimbalances in transcriptional regulation.

Accordingly, provided herein are methods for determining a location ofaccessible genomic DNA in a biological sample. In some instances, themethod includes (a) providing the biological sample on an arraycomprising a plurality of capture probes, wherein a capture probe of theplurality of capture probes comprises: (i) a spatial barcode and (ii) acapture domain; (b) contacting a plurality of splint oligonucleotides tothe biological sample, wherein a portion of a splint oligonucleotide ofthe plurality of splint oligonucleotides hybridizes to a portion of thecapture domain; (c) contacting one or more antibodies, for example,antibodies that recognize a portion of a histone to the biologicalsample, (d) contacting a transposome complex to the biological sample,thereby generating fragmented and tagged genomic DNA (e.g., tagmentedgenomic DNA), wherein the transposome complex includes (i) anantibody-binding moiety (e.g. an antibody-binding protein), (ii) atransposase, (iii) a first transposon end sequence comprising a splintsequence that is substantially complementary to a portion of the splintoligonucleotide, and (iv) the second transposon end sequence comprisinga functional sequence (collectively, (ii), (iii) and (iv) form atransposome); (d) hybridizing a transposon splint sequence of thefragmented genomic DNA to the splint oligonucleotide and ligating thetransposon splint sequence of the fragmented genomic DNA to the capturedomain, thereby generating a capture probe that is ligated to thefragmented genomic DNA; and (e) determining (i) all or part of asequence of the spatial barcode or a complement thereof, and (ii) all orpart of a sequence of the fragmented genomic DNA, or a complementthereof, and using the determined sequences of (i) and (ii) to determineand correlate the location of the accessible genomic DNA in thebiological sample.

Also provided herein are methods for determining and correlating alocation of accessible genomic DNA in a biological sample. In someinstances, the method includes (a) providing the biological sample on anarray comprising a plurality of capture probes, wherein a capture probeof the plurality of capture probes comprises: (i) a spatial barcode and(ii) a capture domain; (b) adding an antibody that binds to a chromatinprotein to the biological sample; (c) binding atransposome-antibody-binding moiety (e.g. an antibody-binding proteinthat contains an antibody-binding moiety, such as protein A, protein G,or a fusion protein thereof) complex (“the complex”) to the antibody,thereby generating fragmented genomic DNA, wherein the complexcomprises: (i) a transposase, (ii) an antibody-binding moiety (e.g. anantibody-binding protein that contains an antibody-binding moiety, suchas protein A, protein G, or a fusion protein thereof), (iii) a firsttransposon end sequence comprising a splint sequence that issubstantially complementary to a portion of the a splintoligonucleotide, and (iv) a second transposon end sequence comprising afunctional sequence; and (d) adding a plurality of splintoligonucleotides to the biological sample, wherein a portion of a splintoligonucleotide of the plurality of splint oligonucleotides hybridizesto a portion of the capture domain; (e) hybridizing the splint sequenceof the fragmented genomic DNA to the splint oligonucleotide andhybridizing the splint oligonucleotide to the capture probe; (f)ligating the splint sequence of the fragmented genomic DNA to thecapture domain; and (g) determining (i) all or part of a sequence of thespatial barcode or a complement thereof, and (ii) all or part of asequence of the fragmented genomic DNA, or a complement thereof, andusing the determined sequences of (i) and (ii) to determine the locationof the accessible genomic DNA in the biological sample.

In any of the methods described herein, binding atransposome-antibody-binding moiety (e.g. an antibody-binding proteinthat contains an antibody-binding moiety, such as protein A, protein G,or a fusion protein thereof) complex (“the complex”) to the antibody,thereby generating fragmented genomic DNA, is performed at roomtemperature. For example, room temperature can include any temperaturebetween 22° C. to 30° C. In some instance, room temperature is 26° C.,27° C., or 28° C.

In any of the methods described herein, binding atransposome-antibody-binding moiety (e.g. an antibody-binding proteinthat contains an antibody-binding moiety, such as protein A, protein G,or a fusion protein thereof) complex (“the complex”) to the antibody,thereby generating fragmented genomic DNA further comprises incubatingthe multi-complex added to the biological sample for one or more hours(e.g. 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 hours), for example, at roomtemperature.

In another embodiment of any of the methods described herein, binding atransposome-antibody-binding moiety (e.g. an antibody-binding proteinthat contains an antibody-binding moiety, such as protein A, protein G,or a fusion protein thereof) complex (“the complex”) to the antibody,thereby generating fragmented genomic DNA further comprises incubatingthe multi-complex added to the biological sample for one or more days(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, or 14 days), forexample, at room temperature.

In some instances, step (d) and the step of determining (i) all or partof a sequence of the spatial barcode or a complement thereof, and (ii)all or part of a sequence of the fragmented genomic DNA, or a complementthereof, and using the determined sequences of (i) and (ii) to determineand correlate the location of the accessible genomic DNA in thebiological sample are performed sequentially. In some instances, step(d) and the step of determining (i) all or part of a sequence of thespatial barcode or a complement thereof, and (ii) all or part of asequence of the fragmented genomic DNA, or a complement thereof, andusing the determined sequences of (i) and (ii) to determine andcorrelate the location of the accessible genomic DNA in the biologicalsample are performed simultaneously. For example, some tagmented DNAfragments can be captured with non-ligated transposon end sequencesstill hybridized. In such examples, the non-ligated transposon endsequences are released after capture of the tagmented DNA. In someinstances, the non-ligated transposon end sequences are released priorto capture by the capture domain.

Also provided herein are methods for determining genomic DNAaccessibility including (a) a biological sample on an array comprising aplurality of capture probes, wherein a capture probe of the plurality ofcapture probes comprises: (i) a spatial barcode and (ii) a capturedomain; (b) contacting the biological sample with one or moreantibodies, for example that will recognize and bind a portion of ahistone, (d) contacting a transposome and/or transposome complex to thebiological sample to insert transposon end sequences into accessiblegenomic DNA, thereby generating fragmented genomic DNA; (c) binding thetransposon end sequences of the fragmented genomic DNA to the capturedomain of the capture probe; (d) releasing one or more transposon endsequences not bound to the capture domain; (e) determining (i) all or aportion of a sequence of the spatial barcode or a complement thereof,and (ii) all or a portion of a sequence of the fragmented genomic DNA,or a complement thereof, and using the determined sequences of (i) and(ii) to determine a location of the accessible genomic DNA in thebiological sample.

In some instances, steps (c) and (d) are performed sequentially. In someinstances, steps (c) and (d) are performed simultaneously. For example,some tagmented DNA fragments can be captured with one or more transposonend sequences still hybridized. In such examples, the one or moretransposon end sequences are released after capture of the tagmentedDNA. In some instances, the one or more transposon end sequences arereleased prior to capture by the capture domain.

In some instances, provided herein are methods for spatial analysis ofnucleic acids (e.g., genomic DNA, mRNA) in a biological sample. In someinstances, an array is provided, wherein the array comprises a pluralityof capture probes. In some instances, the capture probes may be attacheddirectly to the substrate (e.g., an array comprising a substratecomprising a plurality of capture probes). In some instances, thecapture probes may be attached indirectly to the substrate. For example,the capture probes can be attached to features on the substrate. In someinstances, a feature is a bead. In some instances, the capture probescomprise a spatial barcode and a capture domain. In some instances, thecapture probe can be partially double stranded. In some instances, thecapture probe can bind a complementary oligonucleotide. In someinstances, the complementary oligonucleotide (e.g., splintoligonucleotide) can have a single stranded portion. In some instances,the single stranded portion can bind fragmented (e.g., tagmented) DNA.In some instances, a biological sample is treated under conditionssufficient to make nucleic acids in cells of the biological sample(e.g., genomic DNA) accessible to transposon insertion (e.g., taggingthe DNA fragments with transposon ends). In some instances, a transposonend sequence and a transposase enzyme (collectively, a transposome) areprovided to the biological sample such that the transposon end sequencecan be inserted into the genomic DNA of cells present in the biologicalsample. In some instances, the transposase enzyme of the transposomecomplex fragments the genomic DNA and transposon ends are attached tothe ends of the genomic DNA fragments (e.g., “tagmenting”).

In some instances, the biological sample comprising nucleic acids (e.g.,genomic DNA, mRNA) is contacted to the substrate such that a captureprobe can interact with the fragmented and tagged (e.g., tagmented)genomic DNA. In some instances, the biological sample comprising nucleicacids (e.g., genomic DNA, mRNA) is contacted with the substrate suchthat the capture probe can interact with both the tagmented genomic DNAand the mRNA present in the biological sample (e.g., a first captureprobe can bind genomic DNA, a second capture probe can bind mRNA).

In some instances, the location of the capture probe on the substratecan be correlated to a location in the biological sample, therebyspatially determining the location of the tagmented genomic DNA. In someinstances, the location of the capture probe on the substrate can becorrelated to a location in the biological sample, thereby spatiallydetermining the location of the tagmented genomic DNA and mRNA in thebiological sample.

In some instances, where spatial determining the location of analytesincludes a concurrent analysis of different types of analytes from asingle cell or a subpopulation of cells within a biological sample(e.g., a tissue section), an additional layer of spatial information canbe integrated into the genome regulatory interaction maps. In someinstances, the spatial determining of analytes can be done on wholegenomes. In some instances, the spatial profiling can be done on animmobilized biological sample.

Spatial Cleavage Under Targets and Tagmentation (CUT & Tag)

In some instances, the genome-wide chromatic accessibility maps can begenerated by spatial CUT & Tag methods. Without being bound by theory,CUT & Tag uses a transposome that includes a hyperactive Tn5transposase, or another transposase, fused to Protein A (pA-Tn5) orProtein G (pG-Tn5), thereby creating a protein A or protein G, or acombination thereof-transposome complex. The pA-Tn5 or pG-Tn5 complexfurther is loaded with sequencing adapters to bind to a target proteinthat is bound to genomic DNA. Non-limiting examples include those of SEQID NOs: 1-2, and other modified varieties known in the art. (See, forexample, U.S. Pat. Nos. 9,790,476; 10,035,992; and 10,544,403, which areincorporated in their entirety herein). After binding, the complex cuts(i.e., digests) the DNA under the target protein. Released are DNAfragments that are ready for fragment capture on an array describedherein, PCR enrichment, and DNA sequencing. For a detailed descriptionof CUT & Tag, see, for e.g., Kaya-Okur et al. Nature Communications(2019):1930, which is incorporated by reference in its entirety.

In some instances, the genome-wide chromatin accessibility mapsgenerated by spatial CUT & Tag can be used for cell type identification.For example, traditional cell type classification relies on mRNAexpression levels but chromatin accessibility can be more adept atcapturing cell identity. Furthermore, in some instances, correlationsbetween transcriptionally active regions (e.g., open chromatin,accessible) with expression profiles (e.g., expression profiles of mRNA)can be determined in a spatial manner.

Spatial ATAC-seq

In some instances, the genome-wide chromatic accessibility maps can begenerated by spatial ATAC-seq. In some instances, the genome-widechromatin accessibility maps generated by spatial ATAC-seq can be usedfor cell type identification. Without being bound by theory, ATAC-seqmaps a chromatin protein by binding of a specific antibody, and thentethering a Protein A/Micrococcal Nuclease (pA-MNase) fusion protein inpermeabilized cells without cross-linking. MNase is activated byaddition of calcium, and fragments are released into the supernatant forextraction of DNA, library preparation and paired-end sequencing. For adetailed description of ATAC-seq, see, for e.g., Skene and Henikoff(2017). Elife 6, e21856.

In some instances, the genome-wide chromatin accessibility mapsgenerated by spatial ATAC-seq can be used for cell type identification.As indicated above for use with spatial CUT&Tag, traditional cell typeclassification can rely on mRNA expression levels but chromatinaccessibility can be more adept at capturing cell identity. Furthermore,in some instances, correlations between transcriptionally active regions(e.g., open chromatin, accessible) with expression profiles (e.g.,expression profiles of mRNA) can be determined in a spatial manner.

Permeabilizing the Biological Sample

The present disclosure generally describes methods of tagmenting genomicDNA to generate DNA fragments in a biological sample. In some examples,a chemical or enzymatic “pre-permeabilization” of biological samplesimmobilized on a substrate can be employed to make DNA in the biologicalsample accessible to a transposase enzyme (e.g. a transposome or atransposome antibody complex). In some instances, permeabilizing thebiological sample can be a two-step process (e.g., pre-permeabilizationtreatment, followed by a permeabilization treatment). In some instances,permeabilizing the biological sample can be a one-step process (e.g., asingle permeabilization treatment sufficient to permeabilize thecellular and nuclear membranes in the biological sample).

In some instances, pre-permeabilization can include an enzymatic orchemical condition. In some instances, pre-permeabilization can beperformed with an enzyme (e.g., a protease). In some instances, in anon-limiting way, the protease can include trypsin, pepsin, dispase,papain, accuses, or collagenase. In some instances, pre-permeabilizationcan include an enzymatic treatment with pepsin. In some instances,pre-permeabilization can include pepsin in 0.5M acetic acid. In someinstances, pre-permeabilization can include pepsin in Exonuclease-1buffer. In some instances, the pH of the buffer can be acidic. In someinstances, pre-permeabilization can include enzymatic treatment withcollagenase. In some instances, pre-permeabilization can includecollagenase in HBSS buffer. In some instances, the HBSS buffer caninclude bovine serum albumin (BSA). In some instances,pre-permeabilization can last for about 1 to minute to about 20 minutes.In some instances, pre-permeabilization can last for about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, or about 19 minutes. In some instances, pre-permeabilization canlast for about 10 minutes to about one hour. For example, in someinstances, pre-permeabilization can last for about 20, about 30, about40, or about 50 minutes.

In some instances, permeabilizing the biological sample comprises anenzymatic treatment. In some instances, the enzymatic treatment can be apepsin enzyme, or a pepsin-like enzyme treatment. In some instances, theenzymatic treatment can be protease treatment. In some instances,enzymatic treatment can be performed in the presence of reagents. Insome instances, the enzymatic treatment (e.g., pre-permeabilization) caninclude contacting the biological specimen with an acidic solutionincluding a protease enzyme. In some instances, the reagent can be HCl.In some instances, the reagent can be acetic acid. In some instances,the concentration of HCl can be about 100 mM. In some instances, theabout 100 mM HCl can have a pH of around, or about 1.0. In someinstances, the additional reagent can be 0.5M acetic acid, having a pHof around, or about 2.5. It is noted that enzymatic treatment of thebiological sample can have different effects on tagmentation. Forexample, enzymatic treatment with pepsin and 100 mM HCl can result intagmentation of chromatin regardless of chromatin accessibility. In someinstances, enzymatic treatment with pepsin and 0.5M acetic acid canresult in tagmentation of chromatin that can retain a nucleosomalpattern indicative of tagmentation.

In some instances, the enzymatic treatment can comprise contacting thebiological sample with a reaction mixture (e.g., solution) comprising anaspartyl protease (e.g., pepsin) in an acidic buffer, e.g., a bufferwith a pH of about 4.0 or less, such as about 3.0 or less, e.g., about0.5 to about 3.0, or about 1.0 to about 2.5. In some instances, theaspartyl protease is a pepsin enzyme, pepsin-like enzyme, or afunctional equivalent thereof. Thus, any enzyme or combination ofenzymes in the enzyme commission number 3.4.23.1.

In some instances, the enzymatic treatment (e.g., pre-permeabilization)can be performed using collagenase. In some instances, enzymatictreatment with collagenase can provide access to the genomic DNA for thetransposase, transposome, or transposome antibody complex whilepreserving nuclear integrity. In some instances, pre-permeabilization(e.g., enzymatic treatment) with collagenase yields nucleosomal patternsgenerally associated with tagmentation. Collagenases can be isolatedfrom Clostridium histolyticum. In some instances, enzymatic treatmentwith a zinc endopeptidase (e.g., collagenase) with reagents and underconditions suitable for proteolytic activity comprises a bufferedsolution with a pH of about 7.0 to about 8.0 (e.g., about 7.4).Collagenases are zinc endopeptidases and can be inhibited by either EDTAor EGTA, or both. Therefore, in some instances, the biological samplecan be contacted with a zinc endopeptidase (e.g., collagenase) in theabsence of a chelator of divalent cations, (e.g., EDTA, EGTA). In someinstances, it can be useful to stop the zinc endopeptidase (e.g.,collagenase) and the permeabilization step can be stopped (e.g.,inhibited) by contacting the biological sample with a chelator ofdivalent cations (e.g., EDTA, EGTA).

In some instances, the zinc endopeptidase is a collagenase enzyme,collagenase-like enzyme, or a functional equivalent thereof. In suchinstances, any enzyme or combination of enzymes in the enzyme commissionnumber 3.4.23.3 can be used in accordance with materials and methodsdescribed herein. In some instances, the collagenase is one or morecollagenases from the following group, (UniProtKB/Swiss-Prot accessionnumbers): P43153/COLA_CLOPE; P43154/COLA_VIBAL; Q9KRJ0/COLA_VIBCH;Q56696/COLA_VIBPA; Q8D4Y9/COLA_VIBVU; Q9X721/COLG_HATHI;Q46085/COLH_HATHI; Q899Y1/COLT_CLOTE URSTH and functional variants andderivatives thereof (described herein), or a combination thereof.

Methods of permeabilizing biological samples are well known in the art.It will be known to a person skilled in the art that different sourcesof biological samples can be treated with different reagents (e.g.,proteases, RNAses, detergents, buffers) and under different conditions(e.g., pressure, temperature, concentration, pH, time). In someinstances, permeabilizing the biological sample can comprise reagentsand conditions to sufficiently disrupt the cell membrane of thebiological sample to capture nucleic acid (e.g., mRNA). In someinstances, permeabilizing the biological sample can comprise reagentsand conditions to sufficiently disrupt the nuclear membrane of thebiological sample to capture nucleic acid (e.g., genomic DNA). In someinstances, commercially available proteases isolated from their native(e.g., animal, microbial) source can be used. In some instances,proteases produced recombinantly (e.g., bacterial expression system) canbe used. In some instances, pre-permeabilizing and permeabilizing abiological sample can be a one-step process (e.g., enzymatic treatment).In some instances, pre-permeabilizing and permeabilizing a biologicalsample can be a two-step process (e.g., enzymatic treatment, followed bychemical or detergent treatment).

In some instances, the chemical permeabilization conditions comprisecontacting the biological specimen with an alkaline solution, e.g. abuffered solution with a pH of about 8.0 to about 11.0, such as about8.5 to about 10.5 or about 9.0 to about 10.0, e.g. about 9.5. In someinstances, the buffer is a glycine-KOH buffer. Other buffers are knownin the art.

In some instances, a biological sample can be treated with a detergentfollowing an enzymatic treatment (e.g., permeabilization following apre-permeabilization step). Detergents are known in the art. Anysuitable detergent can be used, including, in a non-limiting way NP-40or equivalent, Digitonin, Tween-20, IGEPAL-40 or equivalent, Saponin,SDS, Pitsop2, or combinations thereof. In some instances, a biologicalsample can be treated with other chemicals known to permeabilizecellular membranes. As further exemplified in the examples below,detergents described herein can be used at a concentration of betweenabout 0.01% to about 0.1%. In some instances, detergents describedherein can be used at a concentration of about 0.2%, about 0.3%, about0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, or about 0.9%. Insome instances, detergents described herein can be used at aconcentration of about 1.1% to about 10% or more. In some instances,detergents described herein can be used at a concentration of about 2%,about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, or about 9%,or about 10%.

Additional methods for sample permeabilization are described, forexample, in Jamur et al., Method Mol. Biol. 588:63-66, 2010, the entirecontents of which are incorporated herein by reference. Any suitablemethod for biological sample permeabilization can generally be used inconnection with the biological samples described herein.

Different sources of biological samples can be treated with differentreagents (e.g., proteases, RNAses, detergents, buffers) and underdifferent suitable conditions (e.g., pressure, temperature,concentration, pH, time) to achieve sufficient pre-permeabilization andpermeabilization to capture nucleic acids (e.g., genomic DNA, mRNA).

In some instances, the reaction mixture (e.g., solution) including theproteases described herein can contain other reagents, (e.g., buffer,salt, etc.) sufficient to ensure that the proteases are functional. Forinstance, the reaction mixture can further include an albumin protein,(e.g., BSA). In some instances, the reaction mixture (e.g., solution)including the collagenase enzyme (or functional variant or derivativethereof) includes an albumin protein, (e.g., BSA).

In some instances, there is one or more wash steps betweenpre-permeabilization and permeabilization of a biological sample. Forexample, it may be preferential to wash as much of thepre-permeabilization solution off of the biological sample prior toadding a permeabilization solution. Therefore, in some instances thebiological sample is washed, for example with a SSC wash solution, afterpre-permeabilization to remove the pre-permeabilization reagents andbefore applying permeabilization reagents to the biological sample. Insome instances, the permeabilization solution is removed from thebiological sample prior to the addition of the transposome ortransposome complex reagents for tagmentation of the released genomicDNA. One or more washes may also be performed post permeabilization andpre-tagmentation, for example using a SSC solution. In some instances,there are no wash steps between permeabilization of the biologicalsample and tagmentation of the genomic DNA.

Tagmentation and Spatial Analysis

Transposase enzymes and transposons can be utilized in methods ofspatial genomic analysis. Generally, transposition is the process bywhich a specific genetic sequence (e.g., a transposon sequence) isrelocated from one place in a genome to another. Many transpositionmethods and transposable elements are known in the art (e.g., DNAtransposons, retrotransposons, autonomous transposons, non-autonomoustransposons). One non-limiting example of a transposition event isconservative transposition. Conservative transposition is anon-replicative mode of transposition in which the transposon iscompletely removed from the genome and reintegrated into a new locus,such that the transposon sequence is conserved, (e.g., a conservativetransposition event can be thought of as a “cut and paste” event) (See,e.g., Griffiths A. J., et. al., Mechanism of transposition inprokaryotes. An Introduction to Genetic Analysis (7th Ed.). New York: W.H. Freeman (2000)).

In one example, transposition can occur when a transposase enzyme bindsa sequence flanking the ends of the transposome (e.g., a recognitionsequence, e.g., a mosaic end sequence). A transposome (e.g., atransposition complex) forms and the endogenous DNA can be manipulatedinto a pre-excision complex such that, for example, two transposaseenzymes can interact to form a dimer when the transposase is a Tn5transposase or a tetramer when the transposase is a Mu transposase. Insome instances, when the transposases interact with the DNA, doublestranded breaks are introduced into the DNA resulting in the insertionof the transposon sequence. The transposase enzymes can locate and binda target site in the DNA, create a double stranded break, and insert thetransposon end sequence (See, e.g., Skipper, K. A., et. al., DNAtransposon-based gene vehicles-scenes from an evolutionary drive, JBiomed Sci., 20: 92 (2013) doi:10.1186/1423-0127-20-92). Canonicaltransposases include Tn5. Alternative cut and paste transposases includeTn552 (College, et al, J. BacterioL, 183: 2384-8, 2001; Kirby C et al,Mol. Microbiol, 43: 173-86, 2002), Tyl (Devine & Boeke, Nucleic AcidsRes., 22: 3765-72, 1994 and International Publication WO 95/23875),Transposon Tn7 (Craig, N L, Science. 271: 1512, 1996; Craig, N L, Reviewin: Curr Top Microbiol Immunol, 204:27-48, 1996), Tn/O and IS10(Kleckner N, et al, Curr Top Microbiol Immunol, 204:49-82, 1996),Mariner transposase (Lampe D J, et al, EMBO J., 15: 5470-9, 1996), Tel(Plasterk R H, Curr. Topics Microbiol. Immunol, 204: 125-43, 1996), PElement (Gloor, G B, Methods Mol. Biol, 260: 97-114, 2004), Tn3(Ichikawa & Ohtsubo, J Biol. Chem. 265: 18829-32, 1990), bacterialinsertion sequences (Ohtsubo & Sekine, Curr. Top. Microbiol. Immunol.204: 1-26, 1996), retroviruses (Brown, et al, Proc Natl Acad Sci USA,86:2525-9, 1989), and retrotransposon of yeast (Boeke & Corces, Annu RevMicrobiol. 43:403-34, 1989). More examples include ISS, TnlO, Tn903,IS911, and engineered versions of transposase family enzymes (Zhang etal, (2009) PLoS Genet. 5:e1000689. Epub 2009 Oct. 16; Wilson C. et al(2007) J. Microbiol. Methods 71:332-5).

Transposome-mediated fragmentation and tagging (“tagmentation”) is aprocess of transposase-mediated fragmentation and tagging of DNA. Atransposome is a complex of a transposase enzyme and DNA which comprisesa transposon end sequence (also known as “transposase recognitionsequence” or “mosaic end” (MEs)). In some methods of spatial genomicanalysis, DNA is fragmented in such a manner that a functional sequencesuch as a sequence complementary to a capture domain of a capture probe(e.g., capture domain of a splint oligonucleotide) is inserted into thefragmented DNA (e.g., the fragmented DNA is “tagged”), such that thesequence (e.g. an adapter) can hybridize to the capture probe. In someinstances, the capture probe is present on a substrate. In someinstances, the capture probe (e.g., a capture probe and a splintoligonucleotide) is present on a feature. A transposome dimer, in thecase of the Tn5 transposome, is able to simultaneously fragment DNAbased on its transposon recognition sequences and ligate the transposonsequences from the transposome to the fragmented DNA (e.g., tagmentedDNA). This system has been adapted using hyperactive transposase enzymesand modified DNA molecules (adaptors) comprising MEs to fragment DNA andtag both strands of DNA duplex fragments with functional DNA molecules(e.g., primer binding sites). For instance, the Tn5 transposase may beproduced as purified protein monomers. Tn5 transposase is alsocommercially available (e.g., manufacturer Illumina, Illumina.com,Catalog No. 15027865, TD Tagment DNA Buffer Catalog No. 15027866). Thesecan be subsequently loaded with the oligonucleotides of interest, e.g.,ssDNA oligonucleotides containing MEs (e.g., transposon sequences) forTn5 recognition and additional functional sequences (e.g., Nexteraadapters, e.g., primer binding sites) are annealed to form a dsDNAmosaic end oligonucleotide (MEDS) that is recognized by Tn5 during dimerassembly (e.g., transposome dimerization). In some instances, ahyperactive Tn5 transposase can be loaded with adapters (e.g.,oligonucleotides of interest) which can simultaneously fragment and taga genome with the sequences. As used herein, the term “tagmentation”refers to a step in the Assay for Transposase Accessible Chromatin usingsequencing (ATAC-seq) or a step in the CUT & Tag assay. (See, e.g.,Buenrostro, J. D., Giresi, P. G., Zaba, L. C, Chang, H. Y., Greenleaf,W. J., Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosomeposition, Nature Methods, 10 (12): 1213-1218 (2013)). ATAC-seqidentifies regions of open chromatin using a hyperactive prokaryoticTn5-transposase, which preferentially inserts into accessible chromatinand tags the sites with adaptors (Buenrostro, J. D., et. al.,Transposition of native chromatin for fast and sensitive epigenomicprofiling of open chromatin, DNA-binding proteins and nucleosomeposition. Nat Methods, 10: 1213-1218 (2013)).

As used herein “accessible chromatin” or “open chromatin” or “accessiblegenomic DNA” refers to portions of a genome that are nucleosome-depletedregions that can be bound by proteins and play various roles in nuclearorganization, gene transcription, and are generally consideredtranscriptionally active regions of DNA (Zhang, Q., et al., Genome-wideopen chromatin regions and their effects on the regulation of silkprotein genes in Bombyx mori, Scientific Reports, 7: 12919 (2017).

In some instances, the step of fragmenting the genomic DNA in cells ofthe biological sample comprises contacting the biological samplecontaining the genomic DNA with the transposase enzyme (e.g., atransposome or transposome antibody complex, e.g., a reaction mixture(e.g., solution)) including a transposase, transposome, or transposomeantibody complex), under any suitable conditions. In some instances,such suitable conditions result in the tagmentation of the genomic DNAof cells present in the biological sample. Typical conditions willdepend on the transposase enzyme and/or antibody complexed to thetransposome used and can be determined using routine methods known inthe art. Therefore, suitable conditions can be conditions (e.g., buffer,salt, concentration, pH, temperature, time conditions) under which thetransposase enzyme is functional, e.g., in which the transposase enzymedisplays transposase activity, particularly tagmentation activity, inthe biological sample.

The term “functional”, as used herein in reference to transposaseenzymes, is meant to include instances in which the transposase enzymecan show some reduced activity relative to the activity of thetransposase enzyme in conditions that are optimum for the enzyme, e.g.,in the buffer, salt and temperature conditions recommended by themanufacturer. Thus, the transposase can be considered to be “functional”if it has at least about 50%, e.g., at least about 60%, about 70%, about80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%,about 99%, or about 100%, activity relative to the activity of thetransposase in conditions that are optimum for the transposase enzyme.

In one non-limiting example, the reaction mixture comprises atransposome and/or a transposome antibody complex in a buffered solution(e.g., Tris-acetate) having a pH of about 6.5 to about 8.5, e.g., about7.0 to about 8.0 such as about 7.5. Additionally or alternatively, thereaction mixture can be used at any suitable temperature, such as about10° to about 55° C., e.g., about 10° to about 54°, about 11° to about53°, about 12° to about 52°, about 13° to about 51°, about 14° to about50°, about 15° to about 49°, about 16° to about 48°, about 17° to about47° C., e.g., about 10°, about 12°, about 15°, about 18°, about 20°,about 22°, about 25°, about 28°, about 30°, about 33°, about 35°, aboutor 37° C., preferably about 30° to about 40° C., e.g., about 37° C. Insome instances, the transposome can be contacted with the biologicalsample for about 10 minutes to about one hour. In some instances, thetransposome can be contacted with the biological sample for about 20,about 30, about 40, or about 50 minutes. In some instances, thetransposome can be contacted with the biological sample for about 1 hourto about 4 hours.

In some instances, the transposase enzyme of the transposome complex isa Tn5 transposase, or a functional derivate or variant thereof. (See,e.g., Reznikoff et al, WO 2001/009363, U.S. Pat. Nos. 5,925,545,5,965,443, 7,083,980, and 7,608,434, and Goryshin and Reznikoff, J.Biol. Chem. 273:7367, (1998), which are herein incorporated byreference). In some instances, the Tn5 transposase is a hyper Tn5transposase, or a functional derivate or variate thereof (U.S. Pat. No.9,790,476, incorporated herein by reference). For example, the Tn5transposase can be a fusion protein (e.g., a Tn5 fusion protein). Tn5 isa member of the RNase superfamily of proteins which includes retroviralintegrases. The Tn5 transposon is a composite transposon in which twonear-identical insertion sequences (IS50L and IS50R) flank threeantibiotic resistance genes. Each IS50 contains two inverted 19-bp endsequences (ESs), an outside end (OE) and an inside end (IE). Wild-typeTn5 transposase enzyme is generally inactive (e.g., low transpositionevent activity). However, amino acid substitutions can result inhyperactive variants or derivatives. In one non-limiting example, aminoacid substitution, L372P, substitutes a leucine amino acid for a prolineamino acid which results in an alpha helix break, thus inducing aconformational change to the C-terminal domain. The alpha helix breakseparates the C-terminal domain and N-terminal domain sufficiently topromote higher transposition event activity (See, Reznikoff, W. S., Tn5as a model for understanding DNA transposition, Mol Microbiol, 47(5):1199-1206 (2003)). Other amino acid substitutions resulting inhyperactive Tn5 are known in the art. For example, the improved avidityof the modified transposase enzyme (e.g., modified Tn5 transposaseenzyme) for the repeat sequences for OE termini (class (1) mutation) canbe achieved by providing a lysine residue at amino acid 54, which isglutamic acid in wild-type Tn5 transposase enzyme (See U.S. Pat. No.5,925,545). The mutation strongly alters the preference of the modifiedtransposase enzyme (e.g., modified Tn5 transposase enzyme) for OEtermini, as opposed to IE termini. The higher binding of this mutation,known as EK54, to OE termini results in a transposition rate that isabout 10-fold higher than is seen with wild-type transposase enzyme(e.g., wild type Tn5 transposase enzyme). A similar change at position54 to valine (e.g., EV54) also results in somewhat increasedbinding/transposition for OE termini, as does a threonine to prolinechange at position 47 (e.g., TP47; about 10-fold higher) (See U.S. Pat.No. 5,925,545).

Other examples of modified transposase enzymes (e.g., modified Tn5transposase enzymes) are known. For example, a modified Tn5 transposaseenzyme that differs from wild-type Tn5 transposase enzyme in that itbinds to the repeat sequences of the donor DNA with greater avidity thanwild-type Tn5 transposase enzyme and also is less likely than thewild-type transposase enzyme to assume an inactive multimeric form (U.S.Pat. No. 5,925,545, which is incorporated by reference in its entirety).Furthermore, techniques generally describing introducing anytransposable element (e.g., Tn5) from a donor DNA (e.g., adaptersequence, e.g., Nextera adapters (e.g., top and bottom adapter) into atarget are known in the art. (See, e.g., U.S. Pat. No. 5,925,545).Further study has identified classes of mutations resulting in amodified transposase enzyme (e.g., modified Tn5 transposase enzyme)(See, U.S. Pat. No. 5,965,443, which is incorporated by reference in itsentirety). For example, a modified transposase enzyme (e.g., modifiedTn5 transposase enzyme) with a “class 1 mutation” binds to repeatsequences of donor DNA with greater avidity than wild-type Tn5transposase enzyme. Additionally, a modified transposase enzyme (e.g.,modified Tn5 transposase enzyme) with a “class 2 mutation” is lesslikely than the wild-type Tn5 transposase enzyme to assume an inactivemultimeric form. It has been shown that a modified transposase enzymethat contains both a class 1 and a class 2 mutation can induce at leastabout 100-fold (+10%) more transposition than the wild-type transposaseenzyme, when tested in combination with an in vivo conjugation assay asdescribed by Weinreich, M. D., “Evidence that the cis Preference of theTn5 Transposase is Caused by Nonproductive Multimerization,” Genes andDevelopment 8:2363-2374 (1994), incorporated herein by reference (Seee.g., U.S. Pat. No. 5,965,443). Further, under sufficient conditions,transposition using the modified transposase enzyme (e.g., modified Tn5transposase enzyme) may be higher. A modified transposase enzymecontaining only a class 1 mutation can bind to the repeat sequences withsufficiently greater avidity than the wild-type Tn5 transposase enzymesuch that a Tn5 transposase enzyme induces about 5- to about 50-foldmore transposition than the wild-type transposase enzyme, when measuredin vivo. A modified transposase enzyme containing only a class 2mutation (e.g., a mutation that reduces the Tn5 transposase enzyme fromassuming an inactive form) is sufficiently less likely than thewild-type Tn5 transposase enzyme to assume the multimeric form that sucha Tn5 transposase enzyme also induces about 5- to about 50-fold moretransposition than the wild-type transposase enzyme, when measured invivo (See U.S. Pat. No. 5,965,443)

Other methods of using a modified transposase enzyme (e.g., modified Tn5transposase enzyme are further generally described in U.S. Pat. Nos.5,965,443 and 9,790,476. For example, a modified transposase enzymecould provide selective markers to target DNA, to provide portableregions of homology to a target DNA, to facilitate insertion ofspecialized DNA sequences into target DNA, to provide primer bindingsites or tags for DNA sequencing, or to facilitate production of geneticfusions for gene expression. Studies and protein domain mapping, as wellas, to bring together other desired combinations of DNA sequences(combinatorial genetics) (U.S. Pat. No. 5,965,443).Still other methodsof inserting a transposable element (e.g., transposon) at random orsemi-random locations in chromosomal or extra-chromosomal nucleic acidare known. For example, methods including a step of combining in abiological sample nucleic acid (e.g., genomic DNA) with a synapticcomplex that comprises a Tn5 transposase enzyme complexed with asequence comprising a pair of nucleotide sequences adapted for operablyinteracting with Tn5 transposase enzyme and a transposable element(e.g., transposon) under conditions that mediate transposition eventsinto the genomic DNA. In this method, a synaptic complex can be formedin vitro under conditions that disfavor or prevent synaptic complexesfrom undergoing a transposition event. The frequency of transposition(e.g., transposition events) can be increased by using either ahyperactive transposase enzyme (e.g., a mutant transposase enzyme) or atransposable element (e.g., transposon) that contains sequences welladapted for efficient transposition events in the presence of ahyperactive transposase enzyme (e.g., hyperactive Tn5 transposaseenzyme), or both (U.S. Pat. No. 6,159,736, which is incorporated hereinby reference).

In some instances, the transposome can be fused to (e.g., joined with,linked to, associated with, complexed to) an antibody-binding moiety toform a transposome-antibody binding moiety (e.g. an antibody-bindingprotein that contains an antibody-binding moiety, such as protein A,protein G, or a fusion protein thereof) complex, or a “transposomecomplex”. In some instances, the transposome is fused to protein A. Insome instances, the transposome is fused to protein G. In someinstances, the transposome is fused to a fusion protein of protein A andprotein G (protein A/G). A variety of linkers (e.g. amino acid linkers)that are flexible or rigid can be used. Linkers can include cyclopeptidelinkers, disulfide linkers, and protease-sensitive linkers that arecleavable. In some instances, the transposome is fused to the antibodywith a DDDKEF(GGGGS)₄ linker. Other fusion protein linkers are describedin Chen et al. (2013) Adv. Drug Deliv. Rev. 65(10):1357-1369, which isincorporated by reference in its entirety. The transposome can also befused to the antibody or protein binding moiety post translationallinkers, such as generation of covalent or non-covalent bonds.

An antibody can bind to a target protein that may be associated withchromatin (e.g. bound to), for example a transcription factor, histones,histone PTMs, nucleosomes, ChAPs, and DNA methylation, or any proteinbinding moiety. A protein that binds in a general manner to antibodies,such as protein A, protein G, a fusion protein, a fusion between proteinA and protein G, protein L, protein Y, or functional derivativesthereof, can be used to bind to the antibody that is bound to the targetprotein. The protein biding moiety can include biotin,glutathione-S-transferase (GST), etc. In some instances, the antibodybinding protein can be protein A, or a functional derivative thereof. Insome instances, the antibody binding protein is protein G, or afunctional derivative thereof.

Methods, compositions, and kits for treating nucleic acid, and inparticular, methods and compositions for fragmenting and tagging DNAusing transposon compositions are described in detail in U.S. PatentApplication Publication No. US 2010/0120098, U.S. Patent ApplicationPublication No. US2011/0287435, and Satpathy, A. T., et. al., Massivelyparallel single-cell chromatin landscapes of human immune celldevelopment and intratumoral T-cell exhaustion, Nat Biotechnol., 37,925-936 (2019), the contents of which are herein incorporated byreference in their entireties.

Any transposase enzyme with tagmentation activity, e.g., any transposaseenzyme capable of fragmenting DNA and inserting oligonucleotides (e.g.,adapters, e.g. Nextera index adapters) to the ends of the fragmented(e.g., tagmented) DNA, can be used. In some instances, the transposaseis any transpose capable of conservative transposition. In someinstances, the transposase is a cut and paste transposase. Other kindsof transposase are known in the art and are within the scope of thisdisclosure. For example, suitable transposase enzymes include, withoutlimitation, Mos-1, HyperMu™, Ts-Tn5, Ts-Tn5059, Hermes, Tn7, a Vibharspecies transposase (See e.g., U.S. Patent Application No. 20120301925A1and WO 2015/069374, the contents of which are herein incorporated byreference in their entireties), or any functional variant or derivativeof the previously listed transposase enzymes.

In some instances, a hyperactive variant of the Tn5 transposase enzymeis capable of mediating the fragmentation of double-stranded DNA andligation of synthetic oligonucleotides (e.g., Nextera adapters) at both5′ ends of the DNA in a reaction that takes a short period of time(e.g., about 5 minutes). However, as wild-type end sequences have arelatively low activity, they are sometimes replaced in vitro byhyperactive mosaic end (ME) sequences. A complex of the Tn5 transposasewith 19-bp ME facilitates transposition, provided that the interveningDNA is long enough to bring two of these sequences close together toform an active Tn5 transposase enzyme homodimer.

In some instances, the Tn5 transposase enzyme, or functional variant orderivative thereof, comprises an amino acid sequence having at least 80%sequence identity to SEQ. ID NO. 1. In some instances, the Tn5transposase enzyme, or functional variant or derivative thereof,comprises an amino acid sequence having a sequence identity of at leastabout 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% amino acid sequenceidentity to SEQ ID NO. 1.

In some instances, the transposase enzyme is a Mu transposase enzyme, ora functional variant or derivative thereof.

The adaptors (e.g., Nextera adaptors) in the complex with thetransposase enzyme (e.g., that form part of the transposome, e.g., MEDSdescribed herein) can include partially double strandedoligonucleotides. In some instances, there is a first adapter and asecond adapter. In some instances, the first adapter can be complexedwith a first monomer. In some instances, the second adapter can becomplexed with a second monomer. In some instances, the first monomercomplexed with the first adapter and the second monomer complexed withthe second monomer can be assembled to form a dimer. In some instances,the double stranded portion of the adaptors contains transposon endsequences (e.g., Mosaic End (ME)) sequences. In some instances, thesingle stranded portion of the adaptors (e.g., Nextera index adapters)(5′ overhang) contains the functional domain or sequence to beincorporated in the tagmented DNA. In some instances, the adapters canbe Nextera adapters (e.g., index adapter) (for example, reagentsincluding, Nextera DNA Library Prep Kit for ATAC-seq (no longeravailable), TDE-1 Tagment DNA Enzyme (Catalog No. 15027865), TD TagmentDNA Buffer (Catalog No. 15027866), available from Illumina,Illumina.com). In some instances, the sequence incorporated into thetagmented DNA is a sequence complementary to a capture domain of acapture probe. In some instances, the sequence complementary to thecapture domain of the capture probe is a transposon end sequence. Insuch instances, the functional domain is on the strand of the adaptorthat will be ligated to the capture probe. In other words, thefunctional domain can be located upstream (e.g., 5′ to) the ME sequence,e.g., in the 5′ overhang of the adapter.

The adaptors (e.g., Nextera index adapters, e.g., first and secondadapters) ligated to the tagmented DNA can be any suitable sequence. Forexample, the sequence can be a viral sequence. In some instances, thesequence can be a CRISPR sequence. In some instances, the adaptor (e.g.,oligonucleotides) ligated to the tagmented DNA can be a CRISPR guidesequence. In some instances, the CRISPR guide sequence can target asequence of interest (e.g., genomic locus of interest e.g., genespecific).

In some instances, the ME sequence is a Tn5 transposase recognitionsequence. In some instances, the mosaic end (e.g., ME) sequence is a Mutransposase recognition sequence.

In some instances, a composition comprising a transposase enzyme (e.g.,any transposase enzyme described herein) complexed with adapters (e.g.,first and second adapters complexed with first and second monomers,respectively) comprising transposon end sequences (e.g., mosaic endsequences) is used in a method for spatially tagging nucleic acids in abiological sample. In some instances, a composition comprising atransposase enzyme further comprises a domain that binds to a captureprobe as described herein (e.g., Nextera adapter, e.g., first adapter)and a second adapter is used in a method for spatially tagging nucleicacids of a biological sample, such as any of the methods describedherein.

In some instances, the transposase enzyme can be in the form of atransposome comprising adaptors (MEDS) in which the 5′ overhang can bephosphorylated. In some instances, the adaptors (e.g., Nextera adaptors,e.g., first and second adapters) may be phosphorylated prior to theirassembly with the transposase enzyme to form the transposome. In someinstances, phosphorylation of adaptors can occur when complexed with atransposase enzyme (e.g., phosphorylation in situ in the transposome).

In some instances, the 5′ overhang of the adaptor is not phosphorylatedprior to its assembly in the transposome. In such instances, the 5′overhang can have accessible 5′ hydroxyl groups outside of themosaic-end transposase sequence. In some instances, phosphorylation ofthe 5′ overhang of the assembled transposome complexes can be achievedby exposing these 5′ ends of transposome complexes to a polynucleotidekinase (e.g., T4-polynucleotide kinase (T4-PNK)) in the presence of ATP.

In some instances, tagmenting genomic DNA of the biological sample witha transposome (e.g., any of the transposomes described herein) cancomprise a further step of phosphorylating the 5′ ends of the adaptors(e.g., the 5′ overhangs of the Nextera adaptors, e.g., MEDS) in thetransposome complex.

In some instances, methods provided herein comprise a step of providinga transposome that has been treated to phosphorylate the 5′ ends of theadaptors (e.g., the 5′ overhangs of the Nextera adaptors (e.g., firstand second adapters), e.g., MEDS) in the transposome complex, thusfragmenting the biological sample with a transposome that has beentreated to phosphorylate the 5′ ends of the adaptors in the transposomecomplex.

In some instances, the transposome (e.g., the transposase dimercomplexed with adapters) can be linked to an antibody or peptide with aprotein binding domain. In some instances, the antibody is protein A,protein G, a fusion protein of all or parts of protein A and protein G,or derivatives thereof.

Any suitable enzyme and/or conditions can be used to phosphorylate the5′ ends of the adaptors (e.g., the 5′ overhangs of the adaptors, e.g.,MEDS) in the transposome complex, e.g., T4-PNK or T7-PNK. In someinstances, the phosphorylation reaction can be carried out by contactingthe transposome with a polynucleotide kinase (e.g., T4-PNK or T7-PNK) ina buffered solution (e.g., Tris-HCl, pH about 7.0 to about 8.0, e.g.,about 7.6) at about 20 to about 40° C., e.g., about 25 to about 37° C.,for about 1 to about 60 minutes, e.g., about 5 to about 50, about 10 toabout 40, about 20 to about 30 minutes.

In some instances, gap filling and ligating breaks can be performed onthe fragmented (e.g., tagmented) DNA. For example, the Tn5 transpositionevent results in a 9 base pair gap between an inserted transposon endsequence and the genomic DNA. In some instances, the gap filling isperformed between the inserted transposon end sequence and fragmentedgenomic DNA.

In some instances, the transposon end sequences adjacent to the 9 basepair gap followed by the fragmented genomic DNA are released. In someexamples, the transposon end sequences adjacent to the gap are released(e.g., removed) from the fragmented genomic DNA (e.g., released from thecomplementary transposon end sequence). In some instances, the releasedtransposon end sequences are not ligated to the splint oligonucleotide(e.g., non-ligated transposon end sequences). In some instances, thenon-ligated transposon end sequences are released with a heat gradient.In some instances, the ligated transposon end sequences are ligated tothe capture probe. In some instances, the splint oligonucleotide ishybridized to the capture domain of the capture probe, or a portionthereof, and the remaining transposon end sequence (e.g., ligatedtransposon end sequence). In some instances, a gap filling reaction isperformed. In some instances, gap filling occurs between the splintoligonucleotide and the fragmented genomic DNA. For example, a gapfilling polymerase can fill the gap between the splint oligonucleotideand the fragmented genomic DNA (e.g., a portion of which included thereleased transposon end sequence).

In some instances, the non-ligated transposon end sequences (e.g.,transposon end sequences adjacent to 9 base pair gap) are released witha heat gradient from about 20° C. to about 90° C., from about 25° C. toabout 85° C., from about 30° C. to about 80° C., from about 35° C. toabout 75° C., from about 40° C. to about 75° C., from about 45° C. toabout 75° C., 50° C. to about 75° C., or about 50° C. to about 70° C. Insome instances, releasing the non-ligated transposon end sequencesoccurs at about 25° C., about 26° C., about 27° C., about 28° C., about29° C., about 30° C., about 31° C., about 32° C., about 33° C., about34° C., about 35° C., about 36° C., about 37° C., about 38° C., about39° C., about 40° C., about 41° C., about 42° C., about 43° C., about44° C., about 45° C., about 46° C., about 47° C., about 48° C., about49° C., about 50° C., about 51° C., about 52° C., about 53° C., about54° C., about 55° C., about 56° C., about 57° C., about 58° C., about59° C., about 60° C., about 70° C., about 71° C., about 72° C., about73° C., about 74° C., about 75° C., about 76° C., about 77° C., about78° C., about 79° C., about 80° C., about 81° C., about 82° C., about83° C., about 84° C., about 85° C., about 86° C., about 87° C., about88° C., about 89° C., or about 90° C.

In some instances, releasing the non-ligated transposon end sequences(e.g., transposon end sequences adjacent to the 9 base pair gap) arereleased with a heat gradient for about 10 minutes to about 150 minutes,from about 20 minutes to about 140 minutes, from about 30 minutes toabout 130 minutes, from about 40 minutes to about 120 minutes, fromabout 40 minutes to about 110 minutes, from about 50 minutes to about110 minutes, from about 60 minutes to about 100 minutes, from about 70minutes to about 90 minutes, from about 10 minutes, about 15 minutes,about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes,about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes,about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes,about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes,about 100 minutes, about 105 minutes, about 110 minutes, about 115minutes, about 120 minutes, about 125 minutes, about 130 minutes, about135 minutes, about 140 minutes, about 145 minutes, about 150 minutes,about 155 minutes, about 160 minutes, about 165 minutes, about 170minutes, about 175 minutes, about 180 minutes, about 185 minutes, about190 minutes, about 195 minutes, about 200 minutes, about 205 minutes,about 210 minutes, about 215 minutes, about 220 minutes, about 225minutes, about 230 minutes, about 235 minutes, about 240 minutes, about245 minutes, or about 250 minutes.

In some instances, spatially tagging the genomic DNA can be performed byinsertion of the transposon sequence into the genomic DNA with adaptersdescribed herein. An amplification step can be performed with primers tothe adapters (e.g., inserted adapters into the genomic DNA). Theamplified products can contain accessible genomic DNA which can bespatially tagged by methods described herein.

In some instances, spatially tagging the genomic DNA can be performed bytransposome complexes immobilized on the surface of the substrate. Insome instances, spatially tagging the genomic DNA can be performed bytransposome complexes immobilized on a feature (e.g., a bead). In someinstances, the transposome complexes are assembled prior to adding thebiological sample to the substrate or features. In some instances, thetransposome complexes are assembled after adding the biological sampleto the substrate or features on a substrate. For example, a spatiallybarcoded substrate (e.g., array) can include a plurality of captureprobes that include a Mosaic End sequence (e.g., a transposaserecognition sequence). The Mosaic End sequence can be at the 3′ end ofthe capture probe (e.g., the capture probe is immobilized by its 5′ endand the Mosaic End sequence is at the 3′ most end of the capture probe).The Mosaic End sequence can be a Mosaic End sequence for any of thetransposase enzymes described herein. The Mosaic End sequence (e.g., atransposase recognition sequence) can be hybridized to a reversecomplement sequence (e.g., oligonucleotide). For example, the reversecomplement sequence (e.g., reverse complement to the Mosaic Endsequence) can hybridize to the Mosaic End sequence thereby generating aportion of double stranded DNA on the capture probe. The reversecomplement to the Mosaic End sequence (e.g., oligonucleotide) can beprovided to the spatially barcoded array prior to the biological samplebeing provided to the substrate. In some instances, the reversecomplement to the Mosaic End sequence can be provided after thebiological sample has been provided to the substrate. Transposaseenzymes can be provided to the substrate and assemble at the doublestranded portion of the capture probe (e.g., reverse complementoligonucleotide and the Mosaic End sequence hybridized to each other)thereby generating a transposome complex. For example, a transposomehomodimer can be formed at the double stranded portion of the captureprobe. Additionally, a transposase and antibody, or a transposasecomplexed to an antibody or protein binding moiety) can be provided tothe substrate and assembled at the double stranded portion of thecapture probe (e.g., reverse complement oligonucleotide and the MosaicEnd sequence hybridized to each other) thereby generating a transposomecomplex. A biological sample can be provided to the substrate such thatthe position of the capture probe on the substrate can be correlatedwith a position (e.g., location) in the biological sample. Thetransposome complexes or transposome complex can fragment (e.g.,tagment) and spatially tag the genomic DNA.

In some instances, spatially tagging genomic DNA can be performed byhybridizing a single stranded capture probe to the tagmented DNA. Insome instances the single stranded capture probe can be a degeneratesequence. In some instances, the single stranded capture can be a randomsequence. The single stranded capture probe can have a functionaldomain, a spatial barcode, a unique molecular identifier, a cleavagedomain, or combinations thereof. The single stranded capture probe(e.g., random sequence, degenerate sequence) can non-specificallyhybridize tagmented genomic DNA, thereby spatially capturing thetagmented DNA. Methods for extension reactions are known in the art andany suitable extension reaction method described herein can beperformed.

In other instances, tagmented DNA can be captured by the capture domainof the capture probe by binding (e.g., hybridizing) a poly(A) tailedtagmented DNA (e.g., by including a sequence complementary to a captureprobe on one or more adaptors or recognition sequences (e.g., X1 asshown in FIG. 9 ).

In some instances, after fragmenting the genomic DNA, gap filing (e.g.,no strand displacement) polymerases and ligases can repair gaps andligate breaks in the tagmented DNA. In some instances, a sequencecomplementary to the capture domain can be introduced to the fragmentedDNA. For example, a poly(A) tail can be added to the tagmented DNA, suchthat the capture domain (e.g., poly(T) sequence) of the capture probecan bind (e.g., hybridize) to the poly(A) tailed tagmented DNA (See,e.g., WO 2012/140224, which is incorporated herein by reference). Insome instances, a poly(A) tail is added to the tagmented by a terminaltransferase enzyme. In some instances, the terminal transferase enzymeis a terminal deoxynucleotidyl transferase (TdT), or a mutant variantthereof. TdT is an independent polymerase (e.g., it does not require atemplate molecule) that can catalyze the addition of deoxynucleotides tothe 3′ hydroxyl terminus of DNA molecules. Other template independentpolymerases are known in the art. For example, Polymerase θ, or a mutantvariant thereof, can be used as a terminal transferase enzyme (See,e.g., Kent, T., Polymerase θ is a robust terminal transferase thatoscillates between three different mechanisms during end-joining, eLIFE,5: e13740 doi: 10.7554/eLife.13740, (2016)). Other methods ofintroducing a poly(A) tail are known in the art. In some instances, apoly(A) tail can be introduced to the tagmented DNA by a non-proofreading polymerase. In some instances, a poly(A) tail can be introducedto the fragmented DNA by a polynucleotide kinase.

In some instances, the TDT enzyme will generate tagments with a 3′poly(A) tail, thereby mimicking the poly(A) tail of an mRNA. In someinstances, the capture domain (e.g., poly(T) sequence) of the captureprobe would interact with the poly(A) tail of the mRNA and the generated(e.g., synthesized) poly(A) tail added to the fragmented (e.g.,tagmented) DNA, thereby simultaneously capturing the fragmented DNA(e.g., tagmented DNA) and the mRNA transcript. The generated (e.g.,synthesized) poly(A) tail on the fragmented DNA (e.g., tagmented DNA)could be between about 10 nucleotides to about 30 nucleotides long. Thegenerated (e.g., synthesized) poly(A) tail on the fragmented DNA (e.g.,tagmented DNA) could be about 11, about 12, about 13, about 14, about15, about 16, about 17, about 18, about 19, about 20, about 21, about22, about 23, about 24, about 25, about 26, about 27, about 28, or about29 nucleotides long.

Splint Oligonucleotides

As used herein, the term “splint oligonucleotide” refers to anoligonucleotide that, when hybridized to other polynucleotides, acts asa “splint” (e.g., splint helper probe) to position the polynucleotidesnext to one another so that they can be ligated together. In someinstances, the splint oligonucleotide is DNA or RNA. The splintoligonucleotide can include a nucleotide sequence that is partiallycomplementary to nucleotide sequences from two or more differentoligonucleotides. In some instances, the splint oligonucleotide assistsin ligating a “donor” oligonucleotide and an “acceptor” oligonucleotide.In some instances, an RNA ligase, a DNA ligase, or other ligase can beused to ligate two nucleotide sequences together.

In some instances, the splint oligonucleotide can be between about 10and about 50 nucleotides in length, e.g., between about 10 and about 45,about 10 and about 40, about 10 and about 35, about 10 and about 30,about 10 and about 25, or about 10 and about 20 nucleotides in length.In some instances, the splint oligonucleotide can be between about 15and about 50, about 15 and about 45, about 15 and about 40, about 15 andabout 35, about 15 and about 30, about 15 and about 30, or about 15 andabout 25 nucleotides in length.

In some instances, the fragmented DNA can include a sequence that isadded (e.g., ligated) during fragmentation of the DNA. For example,during a transposition event (e.g., a Tn5 transposition event) anadditional sequence (e.g., transposon end sequences) can be attached(e.g., covalently attached, e.g., via a ligation event) to thefragmented DNA (e.g., fragmented genomic DNA, e.g., tagmented genomicDNA). In some instances, the splint oligonucleotide can have a sequencethat is complementary (e.g., a capture domain) to the fragmented DNA(e.g., fragmented genomic DNA, e.g., fragmented genomic DNA thatincludes a sequence that is added during fragmentation of the DNA, e.g.a first adapter attached during fragmentation of the DNA, e.g., atransposon end sequence) and a sequence that is complementary to thecapture domain of the capture probe. In some instances, the splintoligonucleotide can be viewed as part of the capture probe. For example,the capture probe can be partially double stranded where a portion ofthe capture probe can function as a splint oligonucleotide that binds aportion of the capture probe (e.g., dsDNA portion) and can have a singlestrand portion that can bind (e.g., capture domain) the fragmented DNA(e.g., fragmented genomic DNA e.g., tagmented, e.g., an adapter attachedduring fragmentation of the DNA, e.g., a Nextera adapter). The firstadapter sequence (e.g., the sequence attached to the fragmented DNAcomplementary to the capture domain, e.g., Nextera adapter) can be anysuitable sequence. In some instances, the adapter sequence can bebetween about 15 and 25 nucleotides long. In some instances, the adaptersequence can be about 16, about 17, about 18, about 19, about 20, about21, about 22, about 23, or about 24 nucleotides long.

In some instances, a splint oligonucleotide can include a sequence thatis complementary (e.g., capture domain) to the first adapter attached tothe fragmented DNA (e.g., tagmented DNA). In some instances, the splintoligonucleotide includes a sequence that is not perfectly complementaryto the first adapter (e.g., Nextera adapter) attached to the fragmentedDNA (e.g., tagmented DNA), but is still capable of hybridizing the firstadapter sequence (e.g., sequence complementary to the capture domain)ligated on to the fragmented DNA (e.g., Nextera adapter).

Any of a variety of capture probes having capture domains that hybridizeto a splint oligonucleotide can be used in accordance with materials andmethods described herein. As described herein, a capture domain is adomain on a capture probe capable of hybridizing the splintoligonucleotide to form a partially double stranded capture probe. Forexample, a single stranded capture probe can have a sequencecomplementary (e.g., capture domain) to a portion of the splintoligonucleotide, such that a partially double stranded capture probe isformed with a single stranded portion capable of binding the insertedtransposon end sequences. In some instances, a splint oligonucleotideincludes a sequence that is complementary (e.g., at least partiallycomplementary) to the capture domain of the capture probe.

In some instances, the splint oligonucleotide includes a sequence thatis not perfectly complementary to the capture domain of the captureprobe, but is still capable of hybridizing the capture domain of thecapture probe. In some instances, the splint oligonucleotide canhybridize to both the transposon end sequence (e.g., additional sequenceattached to the tagmented DNA) and the capture domain of the captureprobe via its sequence complementary to the capture domain. In suchinstances, where the splint oligonucleotide can hybridize to both thetransposon end sequence (e.g., Nextera adapter, additional sequenceattached to the fragmented DNA e.g., tagmented DNA), and the capturedomain of the capture probe, the splint oligonucleotide can be viewed aspart of the capture probe.

In some instances, the splint oligonucleotide can have a capture domainthat is homopolymeric. For example, the capture domain can be a poly(T)capture domain.

In some instances, a splint oligonucleotide can facilitate ligation ofthe tagmented DNA and the capture probe. Any variety of suitable ligasesknown in the art or described herein can be used. In some instances, theligase is T4 DNA ligase. In some instances, the ligation reaction canlast for about 1 to about 5 hours. In some instances, the ligationreaction can last for about 2, about 3, or about 4 hours. In someinstances, after ligation, strand displacement polymerization can beperformed. In some instances, a DNA polymerase can be used to performthe strand displacement polymerization. In some instances, the DNApolymerase is DNA polymerase I.

Multiplex Analysis

The present disclosure describes methods for permeabilizing biologicalsamples under conditions sufficient to allow tagmentation of genomicDNA. The genomic DNA, or accessible chromatin, also known as openchromatin, can be tagmented with a transposome complex (e.g. atransposase and an antibody-binding moiety, bound to an antibody boundto, for example, a histone; or a multi-complex of a transposase,antibody-binding moiety, and an antibody, the complex bound to, forexample, a histone). The tagmented genomic DNA can be captured via acapture probe (e.g., a capture probe and a splint oligonucleotide),however, at times it can be useful to simultaneously capture tagmentedgenomic DNA and other nucleic acids (e.g., mRNA). For example,expression profiles of transcripts can be correlated, anti-correlated,or not correlated with open (e.g. accessible) chromatin. Put anotherway, the presence of transcripts can correlate with open chromatin(e.g., accessible chromatin) corresponding to the genes (e.g., genomicDNA) from which the RNA transcripts were transcribed.

The present disclosure also describes methods regarding the simultaneouscapture of tagmented genomic DNA and mRNA or protein on spatiallybarcoded arrays from biological samples (e.g., fresh or frozen tissuesamples). Methods of detecting RNA molecules having poly(A) sequences,proteins, and derivatives of RNA molecules (e.g., RTL products) havebeen described previously in WO 2020/176788 and in U.S. PatentApplication Publication Nos. 2020/0277663, 2021/0285046 and2021/0285036, each of which is incorporated by reference in itsentirety.

For example, multiplex capture (e.g. capturing genomic DNA, DNA, RNAand/or mRNA) can be performed on a spatially barcoded array having aplurality of capture probes immobilized on a substrate surface. Theplurality of capture probes can have substantially the same capturesequence (e.g. a poly(T) capture sequence) and can capture bothtagmented gDNA or mRNA.

Alternatively, multiplex capture can be performed on a spatiallybarcoded array having multiple pluralities of capture probes immobilizedon a substrate surface (e.g. different types of capture probes). In someinstance, tagmented genomic DNA (e.g. tagmented with a transposomecomplex or a multi-complex) can be captured on the array using a splintoligo, as described above. In some instances, the splint oligo iscapable of hybridizing to both a capture probe on an array and to thetagmented DNA. In some instances, the capture probes have capturesequences that are specific to either tagmented DNA or RNA molecules orderivatives of RNA molecules (e.g. RTL products). In some cases, thecapture probes for the RNA molecules or derivatives of RNA molecules(e.g. RTL products) are poly(T) sequences.

In any of the methods described herein, the feature with a plurality ofcapture probes can be on a substrate. The capture probes can havespatial barcodes corresponding to a position (e.g., location) on thesubstrate. In some instances, the capture probes can further have aunique molecular identifier, one or more functional domains, and acleavage domain, or combinations thereof. In some instances, the captureprobe includes a capture domain. In some instances, the capture probecan be a homopolymeric sequence. For example, in a non-limiting way, thehomopolymeric sequence can be a poly(T) sequence. In some instances,nucleic acid (e.g., mRNA) can be captured by the capture domain bybinding (e.g., hybridizing) of poly(A) tails of mRNA transcripts.Tagmented genomic DNA can also be captured by the capture domain of thecapture probe by binding (e.g., hybridizing) a poly(A) tailed tagmentedgenomic DNA (e.g., by including a sequence complementary to a captureprobe on one or more adaptors or recognition sequences (e.g., X1 asshown in FIG. 9 ).

In some instances, after fragmenting the genomic DNA and optionallycapturing RNA molecules having poly(A) sequences or derivatives of RNAmolecules (e.g. via RTL products), gap filing (e.g., no stranddisplacement) polymerases and ligases can repair gaps and ligate breaksin the tagmented DNA. In some instances, a sequence complementary to thecapture domain can be introduced to the fragmented DNA. For example, apoly(A) tail can be added to the tagmented DNA, such that the capturedomain (e.g., poly(T) sequence) of the capture probe can bind (e.g.,hybridize) to the poly(A) tailed tagmented DNA (See, e.g., WO2012/140224, which is incorporated herein by reference). In someinstances, a poly(A) tail is added to the tagmented DNA by a terminaltransferase enzyme. In some instances, the terminal transferase enzymeis a terminal deoxynucleotidyl transferase (TdT), or a mutant variantthereof. TdT is an independent polymerase (e.g., it does not require atemplate molecule) that can catalyze the addition of deoxynucleotides tothe 3′ hydroxyl terminus of DNA molecules. Other template independentpolymerases are known in the art. For example, Polymerase θ, or a mutantvariant thereof, can be used as a terminal transferase enzyme (See,e.g., Kent, T., Polymerase θ is a robust terminal transferase thatoscillates between three different mechanisms during end-joining, eLIFE,5: e13740 doi: 10.7554/eLife.13740, (2016)). Other methods ofintroducing a poly(A) tail are known in the art. In some instances, apoly(A) tail can be introduced to the tagmented DNA by a non-proofreading polymerase. In some instances, a poly(A) tail can be introducedto the fragmented DNA by a polynucleotide kinase. The captured RNAmolecules (e.g. via RTL products) lack gaps and are substantially notaffected by the gap-filling process used on the tagmented DNA.

In some instances, the TDT enzyme will generate tagments with a 3′poly(A) tail, thereby mimicking the poly(A) tail of an mRNA. In someinstances, the capture domain (e.g., poly(T) sequence) of the captureprobe would interact with the poly(A) tail of the mRNA and the generated(e.g., synthesized) poly(A) tail added to the fragmented (e.g.,tagmented) DNA, thereby simultaneously capturing the fragmented DNA(e.g., tagmented DNA) and the mRNA transcript. The generated (e.g.,synthesized) poly(A) tail on the fragmented DNA (e.g., tagmented DNA)could be between about 10 nucleotides to about 30 nucleotides long. Thegenerated (e.g., synthesized) poly(A) tail on the fragmented DNA (e.g.,tagmented DNA) could be about 11, about 12, about 13, about 14, about15, about 16, about 17, about 18, about 19, about 20, about 21, about22, about 23, about 24, about 25, about 26, about 27, about 28, or about29 nucleotides long.

In some instances, provided herein are methods of multiplexing thatcapture both gDNA and RNA. In some instances, in addition to methods ofassociating gDNA (either using a splint oligonucleotide or viahybridization directly), the methods of detecting RNA includehybridizing the analyte (e.g., RNA) or a portion thereof to the capturedomain; and determining (i) all or part of a sequence of the spatialbarcode or a complement thereof, and (ii) all or part of a sequence ofthe analyte, or a complement thereof, and using the determined sequencesof (i) and (ii) to determine the abundance and the location of theanalyte in the biological sample. Methods of RNA capture have beendescribed previously in WO 2020/176788 and in U.S. Patent ApplicationPublication No. 2020/0277663, each of which is incorporated byreference. These methods generally include tissue staining and imaging,cDNA synthesis, second strand synthesis and denaturation, cDNAamplification, library construction, and sequencing.

In some instances, the methods can include determining abundance andlocation of a second analyte in the biological sample by detecting ananalyte derivative such as an RTL product. In some instances, at thesame time as capture of gDNA, the multiplex methods can includecontacting a first templated ligation (RTL) probe and a second RTL probewith the biological sample, wherein the first RTL probe and the secondRTL probe each comprise sequences that are substantially complementaryto adjacent sequences of the second analyte, and wherein the secondprobe comprises a capture probe capture domain that is complementary toall or part of the capture domain; (b) hybridizing the first probe andthe second probe to the second analyte; (c) generating an RTL ligationproduct by ligating the first probe and the second probe; (d) releasingthe RTL ligation product from the second analyte; (e) hybridizing theRTL ligation product to the capture domain; and (f) determining (i) allor part of the sequence of the RTL ligation product bound to the capturedomain, or a complement thereof, and (ii) all or part of the sequence ofthe spatial barcode, or a complement thereof, and using the determinedsequences of (i) and (ii) to identify determine the location of thesecond analyte in the biological sample.

Detection of gDNA using methods described herein can be achievedconcurrently with RNA templated ligation. In some cases, sampleprocessing for determining the location of tagmented gDNA can occurbefore, after, or concurrently with the sample processing fordetermining the location of RNA or RNA-derived products (e.g. via RTLproducts). In some instances, both RTL probes and transposome complexesor RTL probes and multi-complexes can be added to the biological sampleon a spatially barcoded array at the same time. In some instances, RTLprobes are added to the biological sample on a spatially barcoded arraybefore the transposome complexes or multi-complexes are added. In someinstances, RTL probes are added to the biological sample on a spatiallybarcoded array after the transposome complexes or multi-complexes areadded.

In some instances, one of the RTL probes includes a poly(A) sequence, ora complement thereof. In some instances, the poly(A) sequence, or acomplement thereof, is on the 5′ end of one of the RTL probes. In someinstances, the poly(A) sequence, or a complement thereof, is on the 3′end of one of the RTL probes. In some instances, one RTL probe includesa degenerate or UMI sequence. In some instances, the UMI sequence isspecific to a particular target or set of targets. In some instances,the UMI sequence, or a complement thereof, is on the 5′ end of one ofthe RTL probe. In some instances, the UMI sequence, or a complementthereof, is on the 3′ end of one of the RTL probe.

After addition of the RTL probes, RTL probes hybridize to target mRNAand are ligated together. After ligation of the RTL probes, anendonuclease such as RNAse H is added to the sample. RNAse H digestsboth the RNA analyte and the undesirable RNA. In some instances, atleast one of the RTL probes includes a probe capture sequence such as apoly-A sequence, an oligo-d(T) sequence, or a particular capturesequence (in the setting of targeted RNA analysis).

In some instances, the first RTL probe hybridizes to an analyte and asecond RTL probe hybridizes to an analyte in proximity to the first RTLprobe. Hybridization can occur at a target having a sequence that is100% complementary to the probe RTL probe(s). In some instances,hybridization can occur at a target having a sequence that is at least(e.g., at least about) 80%, at least (e.g., at least about) 85%, atleast (e.g., at least about) 90%, at least (e.g., at least about) 95%,at least (e.g., at least about) 96%, at least (e.g., at least about)97%, at least (e.g., at least about) 98%, or at least (e.g., at leastabout) 99% complementary to the RTL probe (s). After hybridization, insome instances, the first RTL probe is extended. After hybridization, insome instances, the second RTL probe is extended. For example, in someinstances a first RTL probe hybridizes to a target sequence upstream ofa second RTL probe, whereas in other instances a first RTL probehybridizes to a target sequence downstream of a second RTL probe.

In some instances, methods disclosed herein include a wash step afterhybridizing the first and the second RTL probes. The wash step removesany unbound probes and can be performed using any technique known in theart. In some instances, a pre-Hybridization buffer is used to wash thesample. In some instances, a phosphate buffer is used. In someinstances, multiple wash steps are performed to remove unbound probes.For example, it is advantageous to decrease the amount of unhybridizedprobes present in a biological sample as they may interfere withdownstream applications and methods.

In some instances, after hybridization of the RTL probes (e.g., thefirst and the second RTL probes) to the target analyte, the RTL probesare ligated together, creating a single ligated probe that iscomplementary to the target analyte. Ligation can be performedenzymatically or chemically, as described herein. For example, the firstand second RTL probes are hybridized to the first and second targetregions of the analyte, and the RTL probes are subjected to a ligationreaction to ligate them together. For example, the probes may besubjected to an enzymatic ligation reaction using a ligase (e.g., T4 RNAligase (Rnl2), a SplintR ligase, or a T4 DNA ligase). See, e.g., ZhangL., et al.; Archaeal RNA ligase from Thermococcus kodakarensis fortemplate dependent ligation RNA Biol. 2017; 14(1): 36-44 for adescription of KOD ligase.

In some instances, adenosine triphosphate (ATP) is added during theligation reaction. DNA ligase-catalyzed sealing of nicked DNA substratesis first activated through ATP hydrolysis, resulting in covalentaddition of an AMP group to the enzyme. After binding to a nicked sitein a DNA duplex, the ligase transfers this AMP to the phosphorylated5′-end at the nick, forming a 5′-5′ pyrophosphate bond. Finally, theligase catalyzes an attack on this pyrophosphate bond by the OH group atthe 3′-end of the nick, thereby sealing it, whereafter ligase and AMPare released. If the ligase detaches from the substrate before the 3′attack, e.g., because of premature AMP reloading of the enzyme, then the5′ AMP is left at the 5′-end, blocking further ligation attempts. Insome instances, ATP is added at a concentration of about 1 μM, about 10μM, about 100 μM, about 1000 μM, or about 10000 μM during the ligationreaction.

In some instances, cofactors that aid in ligation of the RTL probes areadded during the ligation process. In some instances, the cofactorsinclude magnesium ions (Mg²⁺). In some instances, the cofactors includemanganese ions (Mn²⁺). In some instances, Mg²⁺ is added in the form ofMgCl₂. In some instances, Mn²⁺ is added in the form of MnCl₂. In someinstances, the concentration of MgCl₂ is at about 1 mM, at about 10 mM,at about 100 mM, or at about 1000 mM. In some instances, theconcentration of MnCl2 is at about 1 mM, at about 10 mM, at about 100mM, or at about 1000 mM.

In some instances, after ligation of the first and second RTL probes togenerate a ligation product, the ligation product is released from theanalyte. At this stage of the method, (1) the ligation product ishybridized to the analyte, and (2) the gDNA has been tagmented. Torelease the ligation product from the analyte, an endoribonuclease isused. An endoribonuclease such as RNAse H specifically cleaves RNA inRNA:DNA hybrids. Thus, not only does RNAse H cleave the hybridization ofthe ligation product to the analyte (releasing the ligation product),RNAse H also cleaves the undesirable RNA. In some instances, theligation product is released enzymatically. In some instances, anendoribonuclease is used to release the ligation product from theanalyte. In some instances, the endoribonuclease is one or more of RNaseH. In some instances, the RNase H is RNase H1 or RNase H2.

In some instances, after generation of a ligation product from the RTLprobes (e.g., a first RTL probe and second RTL probe), the biologicalsample is permeabilized. In some instances, permeabilization occursusing a protease. In some instances, the protease is an endopeptidase.Endopeptidases that can be used include but are not limited to trypsin,chymotrypsin, elastase, thermolysin, pepsin, clostripan, glutamylendopeptidase (GluC), ArgC, peptidyl-asp endopeptidase (ApsN),endopeptidase LysC and endopeptidase LysN. In some instances, theendopeptidase is pepsin. In some instances, permeabilization isperformed using proteinase K.

In some instances, the ligation product includes a capture probe bindingdomain, which can hybridize to a capture probe (e.g., a capture probeimmobilized, directly or indirectly, on a substrate). In some instances,methods provided herein include contacting a biological sample with asubstrate, wherein the capture probe is affixed to the substrate (e.g.,immobilized to the substrate, directly or indirectly). In someinstances, the capture probe includes a spatial barcode and the capturedomain. In some instances, the capture probe binding domain of theligation product binds (e.g., hybridizes) to the capture domain. Afterhybridization of the ligation product to the capture probe, the ligationproduct is extended at the 3′ end to make a copy of the additionalcomponents (e.g., the spatial barcode) of the capture probe. In someinstances, methods of ligation product capture as provided hereininclude permeabilization of the biological sample such that the captureprobe can more easily hybridize to the ligation product (i.e., comparedto no permeabilization). In some instances, reverse transcription (RT)reagents can be added to permeabilized biological samples. Incubationwith the RT reagents provide for the extension of the capture probeusing the ligation product as a template, as well as the extension ofthe ligation product using the capture probe as a template therebyproducing nucleic acid molecules that comprise target analyte sequences,or complements thereof, and sequences of the capture probe, orcomplements thereof such as spatial barcodes, functional sequences,UMIs, etc.

The resulting nucleic acid molecule can be denatured from the captureprobe template and transferred (e.g., to a clean tube) foramplification, and/or library construction as described herein. Thespatially-barcoded, full-length nucleic acid molecule can be amplifiedvia PCR prior to library construction. The nucleic acid molecule canthen be enzymatically fragmented and size-selected in order to optimizethe amplicon size. P5, P7, i7, and i5 can be used as sample indexes, andTruSeq Read 2 can be added via End Repair, A-tailing, Adaptor Ligation,and PCR. The fragments can then be sequenced using paired-end sequencingusing TruSeq Read 1 and TruSeq Read 2 as sequencing primer sites.

Post-hybridization steps are also provided in Stahl P. L., et al.,Visualization and analysis of gene expression in tissue sections byspatial transcriptomics Science, vol. 353, 6294, pp. 78-82 (2016), whichin incorporated herein by reference).

In any of the methods described herein, the tissue sample is aformalin-fixed, paraffin-embedded (FFPE) tissue sample, a fresh tissuesample, or a frozen tissue sample. In some instances, the tissue sampleis the FFPE tissue sample. In some instances, the biological sample isdeparaffinized. Deparaffinization can be achieved using any method knownin the art. For example, in some instances, the biological samples istreated with a series of washes that include xylene and variousconcentrations of ethanol. In some instances, methods ofdeparaffinization include treatment of xylene (e.g., three washes at 5minutes each). In some instances, the methods further include treatmentwith ethanol (e.g., 100% ethanol, two washes 10 minutes each; 95%ethanol, two washes 10 minutes each; 70% ethanol, two washes 10 minuteseach; 50% ethanol, two washes 10 minutes each). In some instances, afterethanol washes, the biological sample can be washed with deionized water(e.g., two washes for 5 minutes each). It is appreciated that oneskilled in the art can adjust these methods to optimizedeparaffinization.

In some instances, the biological sample is decrosslinked. In someinstances, the biological sample is decrosslinked in a solutioncontaining TE buffer (comprising Tris and EDTA). In some instances, theTE buffer is basic (e.g., at a pH of about 9). In some instances,decrosslinking occurs at about 50° C. to about 80° C. In some instances,decrosslinking occurs at about 70° C. In some instances, decrosslinkingoccurs for about 1 hour at 70° C. Just prior to decrosslinking, thebiological sample can be treated with an acid (e.g., 0.1M HCl for about1 minute). After the decrosslinking step, the biological sample can bewashed (e.g., with 1×PBST).

Methods of Making a Protein A/G Transposome Complex

Protein A is a 40-60 kDA surface protein found in Staphylococcus aureuscell walls. Its ability to bind immunoglobulins and IgG proteins, theheavy chain within the Fc region of most immunoglobulins, from manymammalian species makes it a useful protein for complexing withantibodies. It has the ability to interact and associate with a numberof antibodies to create multiple options forprotein-transposome-antibody complexing. Protein G is an immunoglobulinbinding protein found in Streptococcal bacteria, but differs fromProtein A in its binding properties. Protein G is a cell surface bindingprotein of 40-65 kDa, that binds to the Gav and Fc region of multipleantibodies, and indeed it finds utility in purifying antibodies becauseof its wide ability for antibody binding. Associating either protein A,protein G, or a protein A/G fusion protein with the transposase tocreate a protein A/G-Tn5 transposome can include generating a contiguousnucleic acid sequence via, e.g., standard cloning techniques. Adaptersto be inserted into the transposome are added by complexing the proteinA/G transposase complex with the associated transposon sequences asknown in the art.

For example, Kaya-Okur et al (2019, Nature Communications 10:1930CUT&Tag for efficient epigenomic profiling of small samples and singlecells (incorporated herein by reference), provides a method forgenerating a protein A-Tn5 transposome complex by first generating aC-terminal fusion protein with protein A separated from the transposaseby a 26 amino acid long flexible peptide linker, followed by transposomegeneration with the generated protein A-transposase fusion proteincomplexed with the transposon mosaic ends. Another method for generatinga protein A-Tn5 transposome complex can be found in Bartosovic et al(2021, Nat Biotech, doi.org/10.1038/s41587-021-00869-9, incorporatedherein by reference). Commercially available protein A-Tn5 transposasecomplexes for generating a protein A fused transposomes are available,for example from Vazyme (Hyperactive pA-Tn5 Transposase for CUT&Tag).

The present disclosure is not limited by the methods in which a proteinA or protein G-transposome complexes are generated, further the presentdisclosure is not limited by the methods of generating anantibody-transposome complex, where the antibody-transposome complexcomprises an antibody bound to protein A or protein G of a proteinA-transposome complex or a protein G-transposome complex.

Compositions and Kits

Also provided herein are compositions including capture probes,transposome complexes, tagmented DNA, splint oligonucleotides, and oneor more polymerases. In some instances, a splint oligonucleotide ishybridized to the capture domain of a capture probe. In some instances,a splint oligonucleotide is hybridized to a transposon end sequence offragmented (e.g., tagmented) genomic DNA. In some instances, a splintoligonucleotide is hybridized to the captured domain of a capture probeand a transposon end sequence of fragmented genomic DNA. In someinstances, the composition comprises one or more transposon endsequences. In some instances, one or more transposon end sequences areligated to the capture probe. In some instances, one or more transposonsequences are released from the fragmented DNA either before or afterligation of the fragmented genomic DNA to the capture probe. In someinstances, the composition includes a ligase (e.g., T4 DNA ligase). Insome instances, the composition includes a gap filling polymerase. Insome instances, the composition includes a DNA polymerase.

In some instances, the capture domain of the capture probe binds thetransposon end (e.g., without the facilitation by a splintoligonucleotide). In some instances, the composition includes astrand-displacing polymerase. In some instances, the compositionincludes a gap filling polymerase.

In some instances, the composition includes an array comprising aplurality of capture probes. In some instances, a capture probe of theplurality of capture probes include a spatial barcode and a capturedomain.

In some instances, the composition includes a transposome. In someinstances, the composition includes a protein bindingprotein-transposome complex. In some instance, the transposome complexincludes a protein binding moiety, a transposase, a first transposon endsequence comprising a splint sequence that is substantiallycomplementary to a portion of a splint oligonucleotide, and a secondtransposon end sequence.

Also provided herein are kits that include one or more reagents todetect one or more analytes described herein. In some instances, the kitincludes an array comprising a plurality of capture probes comprising aspatial barcode and a capture domain. In some instances, the kitincludes a transposome complex comprising an antibody, a transposase, afirst transposon end sequence comprising a splint sequence that issubstantially complementary to a portion of a splint oligonucleotide,and a second transposon end sequence comprising a functional sequence.In some instances, the kit includes a plurality of probes (e.g., RDprobes, a first RTL probe, a second RTL probe, one or more spanningprobes, and/or a third oligonucleotide).

A non-limiting example of a kit used to perform any of the methodsdescribed herein includes: (a) an array comprising a plurality ofcapture probes, wherein a capture probe of the plurality of captureprobes comprises: (i) a spatial barcode and (ii) a capture domain; (b) atransposome complex comprising: (i) an antibody, (ii) a transposase,(iii) a first transposon end sequence comprising a splint sequence thatis substantially complementary to a portion of a splint oligonucleotide,and (iv) a second transposon end sequence comprising a functionalsequence; and (c) instructions for performing any one of the methodsdescribed herein.

In some instances of any of the kits described herein, the kit includesa second RTL probe that includes a preadenylated phosphate group at its5′ end and a first RTL probe comprising at least two ribonucleic acidbases at the 3′ end.

qPCR and Analysis

Also provided herein are methods and materials for quantifying captureefficiency. In some instances, quantification of capture efficiencyincludes quantification of captured fragments (e.g., genomic DNAfragments, e.g., tagmented DNA fragments) from any of the spatialanalysis methods described herein. In some instances, quantificationincludes PCR, qPCR, electrophoresis, capillary electrophoresis,fluorescence spectroscopy and/or UV spectrophotometry. In someinstances, qPCR includes intercalating fluorescent dyes (e.g., SYBRgreen) and/or fluorescent labeled-probes (e.g., without limitation,Taqman probes or PrimeTime probes). In some instances, a NGS libraryquantification kit is used for quantification. For example,quantification can be performed using a KAPA library quantification kit(KAPA Biosystems), qPCR NGS Library Quantification Kit (Agilent),GeneRead Library Quant System (Qiagen), and/or PerfeCTa NGSQuantification Kit (Quantabio). In some instances that use qPCR forquantification, qPCR can include, without limitation, digital PCR,droplet digital (ddPCR), and ddPCR-Tail. In some instances that useelectrophoresis for quantification, electrophoresis can include, withoutlimitation, automated electrophoresis (e.g., TapeStation System,Agilent, and/or Bioanalzyer, Agilent) and capillary electrophoresis(e.g., Fragment Analyzer, Applied Biosystems). In some instances thatuse spectroscopy for quantification, the spectroscopy can include,without limitation, fluorescence spectroscopy (e.g., Qubit, ThermoFisher). In some instances, NGS can be used to quantify captureefficiency.

In some instances, quantitative PCR (qPCR) is performed on the capturedtagments. In some instances, the fragmented (e.g., tagmented) DNA isamplified, by any method described herein, before capture. For example,after capture of the fragmented DNA (e.g., tagmented DNA), ligation andstrand displacement hybridization qPCR can be performed.

Methods of staining a biological sample (e.g., immunofluorescence,immunohistochemistry, H&E) are known in the art and are provided herein.In some instances, the biological sample can be imaged.

EXAMPLES Example 1. Assessing Accessible Chromatin in a Spatial Contextwith a Transposome Complex Workflow

An antibody binding moiety-transposome complex (FIG. 9 ) is constructedof a Tn5 dimerized transposase linked to a protein A or protein G. TheTn5 transposase is loaded with transposon sequences that contain 19-bpmosaic ends (ME), optionally capture domains, and primer sequences. Thetransposase and complexed transposons collectively form a transposome.

An antibody that binds, for example, a histone is applied to thebiological sample and the antibody is allowed to bind to its targetantigen, The transposome complex is applied to a biological sample whichis located on a spatial array, and the protein A or protein G moietybinds to the antibody that is bound to the target protein (such as ahistone). The genomic DNA, or open chromatin, of the cell of the tissuecan undergo tagmentation, mediated by the transposome complex. The openchromatin or open genomic DNA that may be present between histones isavailable for a potential transpositional event as the transposase cutsthe genomic DNA and inserts the transposon ends into the cut genomic DNA(FIG. 10 ). As such, the fragmented genomic DNA includes on either endone of the transposon end sequences (e.g., R1 or X1 related ends).

The resulting tagmented gDNA in the biological sample can interact withcapture probes on the spatial array, each capture probe having a capturedomain and a spatial barcode, wherein the tagmented gDNA is attached tothe capture probe using a splint oligonucleotide (FIG. 11 ). Theresulting nucleic acid comprising the capture probe, splintoligonucleotide (“splint oligo” in FIG. 11 ), and tagmented gDNA areligated and a gap-filling extension step to fill the gap betweentagmented gDNA and the capture probe is performed, as well as gapsgenerated by the tagmentation process (e.g., at the 3′ end of thecapture domain). As a result, a primary strand attached to the captureprobe and immobilized on the array is generated, the primary strandincludes the capture probe and a sequence comprising the tagmented gDNAor a complement thereof. Attached to the primary strand is a secondstrand full-length product that is released by heating, and used e.g.,as a PCR template. A library can be generated from the released productwhich can be sequenced and the spatial location of the sequenced productcan be determined and correlated with a location in the biologicaltissue sample by determining all or part of the sequence of the spatialbarcode on the capture probe and all or part of the tagmented gDNAsequence.

Example 2. Gap-Filling Between the Tagmented DNA and the Capture Probe

After the tagmented gDNA of Example 1 interacts with capture probes onthe spatial array, with each capture probe having a capture domain and aspatial barcode, the tagmented gDNA is attached to the capture probeusing a splint oligonucleotide (FIG. 11 ). The resulting nucleic acidcomprising the capture probe, splint oligonucleotide (“splint oligo” inFIG. 11 ), and tagmented gDNA can have gaps, or spaces of non-ligatednucleic acids, between the end of one oligonucleotide and the beginningof another. For example, there may be a gap between the splint oligo andthe X1 mosaic end, and/or between the X1 mosaic end and the gDNA.

The gaps can be filled, for example, by a “gap-filling” step whichincludes incorporation of one or more nucleic acids by a polymerase,based on the nucleic acid sequence of a template nucleic acid molecule,spanning a distance between the two nucleic acid molecules of interest,followed by ligation of the two now adjacent ends. As such, anon-interrupted nucleic acid is produced that contains at least the UMI,capture sequence, transposon sequences with mosaic ends, genomic DNA,another transposon sequence, and sequencing primer binding sites (FIG.11 ).

Example 3. Assessing Accessible Chromatin in a Spatial Context with aMulti-Complex Workflow

A multi-complex of a transposome, an antibody-binding moiety, and anantibody complex (FIG. 10 ) is constructed of a Tn5 transposome linkedto a protein A or protein G, which is bound to an antibody that canbind, for example, histones. The Tn5 transposase is loaded withtransposon sequences that contain 19-bp mosaic ends (ME) (e.g.,transposome collectively), optionally capture domains, and primersequences. The transposase, complexed transposons, and antibody iscollectively referred to as a multi-complex.

The multi-complex with the antibody that binds, for example, a histone,is applied to the biological sample located on a spatial array. Theantibody of the multi-complex binds to its target antigen, such as atarget protein (e.g., histone). The genomic DNA, or open chromatin, ofthe cell of the biological sample, such as a tissue, can undergotagmentation, mediated by the transposome of the multi-complex. The openchromatin or open genomic DNA that may be present between histones isavailable for a potential transpositional event as the transposase cutsthe genomic DNA and inserts the transposon ends into the cut genomic DNA(FIG. 10 ). As such, the fragmented genomic DNA includes, on either end,transposon end sequences (e.g. R1 or X1 related ends).

The resulting tagmented gDNA in the biological sample can interact withcapture probes on the spatial array, each capture probe having a capturedomain and a spatial barcode, wherein the tagmented gDNA is attached tothe capture probe using a splint oligonucleotide, as described inExample 1 (FIG. 11 ). A library can be generated from the releasedproduct which can be sequenced and the spatial location of the sequencedproduct can be determined and correlated with a location in thebiological tissue sample by determining all or part of the sequence ofthe spatial barcode on the capture probe and all or part of thetagmented gDNA sequence.

Sequence Listing Tn5 Transposase SEQ ID NO: 1MITSALHRAADWAKSVFSSAALGDPRRTARLVNVA AQLAKYSGKSITISSEGSEAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYRH QVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESGKWLAAAATSRLR MGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLYLYDHLKNQPELGGYQISIPQKG VVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPPKGETPLKWLLLTSEPVESLAQALRV IDIYTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILSFVAVRLLQLRESFTLPQALRAQGLLKEA EHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEA LQSKLDGFLAAKDLMAQGIKITn5 Transposase (UniProtKB/Swiss-Prot: Q46731.1) SEQ ID NO: 2MITSALHRAADWAKSVFSSAALGDPRRTARLVNVA AQLAKYSGKSITISSEGSEAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYRH QVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESGKWLAAAATSRLR MGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLYLYDHLKNQPELGGYQISIPQKG VVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPPKGETPLKWLLLTSEPVESLAQALRV IDIYTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILSFVAVRLLQLRESFTLPQALRAQGLLKEA EHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEA LQSKLDGFLAAKDLMAQGIKI

1. A method for determining location of accessible genomic DNA in abiological sample, the method comprising: (a) providing one or moreantibodies to the biological sample, wherein an antibody of the one ormore antibodies binds to a chromatin protein in the biological sample;(b) binding a transposome-antibody-binding moiety complex to the one ormore antibodies, wherein the transposome-antibody-binding moiety complexcomprises: (i) a transposase, (ii) an antibody-binding moiety, and (iii)one or more transposon end sequences; (c) generating fragmented genomicDNA comprising the one or more transposon end sequences; (d) applying afirst microfluidic device having multiple first addressing channels tothe biological sample, wherein a first addressing channel identifies afirst area in the biological sample; (e) delivering a first probethrough the first addressing channel to the first area in the biologicalsample, wherein the first probe comprises (i) a first ligation regionfor ligation to a transposon end sequence of the one or more transposonend sequences, (ii) a first address tag that identifies the first areain the biological sample, and (iii) a second ligation region; (f)applying a second microfluidic device having multiple second addressingchannels to the biological sample, wherein a second addressing channelidentifies a second area in the biological sample that intersects withthe first area; and (g) delivering a second probe through the secondaddressing channel to the second area in the biological sample, whereinthe second probe comprises: (i) a second address tag that identifies thesecond area in the biological sample and (ii) a third ligation region,wherein the second probe is coupled to the first probe at the secondligation region and the third ligation region at an intersection betweenthe first area and the second area, thereby generating a ligationproduct, and wherein the first and second address tags are used toidentify the location of the accessible genomic DNA in the biologicalsample.
 2. The method of claim 1, wherein the one or more antibodiescomprises the antibody and a secondary antibody that binds to theantibody.
 3. The method of claim 2, wherein thetransposome-antibody-binding moiety complex binds to the secondaryantibody.
 4. The method of claim 1, wherein steps (a) and (b) areperformed at substantially the same time, and wherein the antibody andthe transposome-antibody-binding moiety complex are combined to form amulti-complex.
 5. The method of claim 1, wherein step (a), through step(e) are performed at substantially the same time.
 6. The method of claim1, wherein the antibody-binding moiety is protein A, protein G, orfunctional derivatives thereof.
 7. The method of claim 1, wherein thetransposase is a Tn5 transposase enzyme, a Mu transposase enzyme, a Tn7transposase enzyme, a Vibhar species transposase, or functionalderivatives thereof.
 8. The method of claim 1, wherein the biologicalsample is permeabilized prior to step (b), wherein permeabilization ischemical permeabilization or enzymatic permeabilization, and wherein thechemical permeabilization utilizes a reagent comprising a detergent,optionally wherein the detergent is one or more of NP-40,polysorbate-20, and digitonin.
 9. The method of claim 8, wherein theenzymatic permeabilization utilizes a reagent comprising a protease ofthe group consisting of a pepsin, a collagenase, a proteinase K, orcombinations thereof.
 10. The method of claim 1, wherein the transposonend sequence is inserted into the accessible genomic DNA.
 11. The methodof claim 1, further comprising (i) coupling the first probe to thetransposon end sequence or (ii) coupling the second probe to the firstprobe between the second ligation region and the third ligation regionvia ligation.
 12. The method of claim 11, wherein the ligation comprisesenzymatic ligation, wherein the enzymatic ligation utilizes a ligaseselected from the group consisting of a PBCV-1 DNA ligase, a Chlorellavirus DNA ligase, a single-stranded DNA ligase, and a T4 DNA ligase. 13.The method of claim 11, wherein the ligation comprises use of one ormore splint oligonucleotides comprising: (i) a sequence that hybridizesto a portion of the transposon end sequence; and (ii) a sequence thathybridizes to a portion of the first probe; or (iii) a sequence thathybridizes to a portion of the first probe; and (iv) a sequence thathybridizes to a portion of the second probe.
 14. The method of claim 11,wherein the ligation comprises chemical ligation.
 15. The method ofclaim 1, wherein the transposon end sequence is at least about 10nucleotides to about 50 nucleotides long.
 16. The method of claim 1,wherein the multiple first addressing channels or multiple secondaddressing channels is n addressing channels, wherein n is an integerbetween about 5 and about
 100. 17. The method of claim 1, wherein eachof the multiple first addressing channels and multiple second addressingchannels has a width of about 5 μm to about 500 μm.
 18. The method ofclaim 1, wherein each of the multiple first addressing channels andmultiple second addressing channels has a depth of about 5 μm to about500 μm.
 19. The method of claim 1, wherein each of the multiple firstaddressing channels and multiple second addressing channels has adistance from other multiple addressing channels of about 5 μm to about2 mm.
 20. The method of claim 1, wherein the first probe and/or thesecond probe further comprises a variable tag region, a sequencingadaptor, or a combination thereof.
 21. The method of claim 1, whereinthe first probe and/or the second probe comprises a nucleic acidsequence.
 22. The method of claim 21, wherein the nucleic acid sequencecomprises DNA.
 23. The method of claim 1, further comprising amplifyingthe ligation product.
 24. The method of claim 23, wherein the amplifyingis performed by polymerase chain reaction.
 25. The method of claim 1,wherein the biological sample is a fixed sample, a frozen sample, afresh sample, or a fresh frozen sample.
 26. The method of claim 25,wherein the fixed sample is a formalin fixed paraffin embedded sample.27. The method of claim 1, wherein (i) the first and second addresstags, and (ii) all or part of the ligation product, or a complementthereof are sequenced to determine the location of the accessiblegenomic DNA.
 28. The method of claim 1, further comprising determining alocation of one or more additional accessible genomic DNA in thebiological sample, wherein the one or more additional accessible genomicDNA is an integer between 100 and 30,000.
 29. The method of claim 1,further comprising staining and imaging the biological sample, whereinthe staining is selected from immunohistochemistry, immunofluorescence,hematoxylin, eosin, or a combination thereof.
 30. A compositioncomprising: (a) a transposome complex; (b) one or more antibodiescomprising (i) an antibody or (ii) the antibody and a secondary antibodythat binds to the antibody; and (c) an antibody binding moiety, whereinthe antibody binding moiety is simultaneously bound to the one or moreantibodies and the transposome complex; and wherein the antibody isbound, directly or indirectly, to a nuclear protein.